Extended tethering approach for rapid identification of ligands

ABSTRACT

The invention concerns a method for rapid identification and characterization of binding partners for a target molecule, and for providing binding partners with improved binding affinity. More specifically, the invention concerns an improved tethering method for the rapid identification of at least two binding partners that bind near one another to a target molecule.

This application is a continuation-in-part and claims priority under 35U.S.C. § 1.19(e) of U.S. Provisional Application No. 60/252,294 filed onNov. 21, 2000 and U.S. Provisional Application No. 60/310,725 filed onAug. 7, 2001.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to a method for rapididentification and characterization of binding partners for a targetmolecule, and for providing modified binding partners with improvedbinding affinity. More specifically, the invention concerns an improvedtethering method for the rapid identification of small moleculefragments that bind near one another on a target molecule. The method isparticularly suitable for rapid identification of small molecule ligandsthat bind weakly near sites of interest through a preformed linker on atarget biological molecule (TBM), such as a polypeptide or othermacromolecule, to produce higher affinity compounds.

2. Description of the Related Art

The drug discovery process usually begins with massive screening ofcompound libraries (typically hundreds of thousands of members) toidentify modest affinity leads (K_(d) ˜1 to 10 μM). Although sometargets are well suited for this screening process, most are problematicbecause moderate affinity leads are difficult to obtain. Identifying andsubsequently optimizing weaker binding compounds would improve thesuccess rate, but screening at high concentrations is generallyimpractical because of compound insolubility and assay artifacts.Moreover, the typical screening process does not target specific sitesfor drug design, only those sites for which a high-throughput assay isavailable. Finally, many traditional screening methods rely oninhibition assays that are often subject to artifacts caused by reactivechemical species or denaturants.

Erlanson et al., Proc. Nat. Acad Sci. USA 97:9367-9372 (2000), haverecently reported a new strategy, called “tethering”, to rapidly andreliably identify small (˜250 Da) soluble drug fragments that bind withlow affinity to a specifically targeted site on a protein or othermacromolecule, using an intermediary disulfide “tether.” According tothis approach, a library of disulfide-containing molecules is allowed toreact with a cysteine-containing target protein under partially reducingconditions that promote rapid thiol exchange. If a molecule has evenweak affinity for the target protein, the disulfide bond (“tether”)linking the molecule to the target protein will be entropicallystabilized. The disulfide-tethered fragments can then be identified by avariety of methods, including mass spectrometry (MS), and their affinityimproved by traditional approaches upon removal of the disulfide tether.See also PCT Publication No. WO 00/00823, published on Jan. 6, 2000.

Although the tethering approach of Erlanson et al. represents asignificant advance in the rapid identification of small low-affinityligands, and is a powerful tool for generating drug leads, there is aneed for further improved methods to facilitate the rational design ofdrug candidates.

SUMMARY OF THE INVENTION

The present invention describes a strategy to rapidly and reliablyidentify ligands that have intrinsic binding affinity for differentsites on a target molecule by using an extended tethering approach. Thisapproach is based on the design of a Small Molecule Extender (SME) thatis tethered, via a reversible or irreversible covalent bond, to a TargetMolecule (TM) at or near a first site of interest, and has a chemicallyreactive group reactive with small organic molecules to be screened foraffinity to a second site of interest on the TM. Accordingly, the SME isused for screening a plurality of ligand candidates to identify a ligandthat has intrinsic binding affinity for a second site of interest on theTM. If desired, further SME's can be designed based on theidentification of the ligand with binding affinity for the second siteof interest, and the screening can be repeated to identify furtherligands having intrinsic binding affinity for the same or other site(s)of interest on the same or related TM's.

One aspect of the invention concerns the design of a Small MoleculeExtender (SME). In this aspect, the invention concerns a processcomprising:

-   -   (i) contacting a Target Molecule (TM) having a first and a        second site of interest, and containing or modified to contain a        reactive nucleophile or electrophile at or near the first site        of interest with a plurality of first small organic ligand        candidates, the candidates having a functional group reactive        with the nucleophile or electrophile, under conditions such that        a reversible covalent bond is formed between the nucleophile or        electrophile and a candidate that has affinity for the first        site of interest, to form a TM-first ligand complex;    -   (ii) identifying the first ligand from the complex of (i); and    -   (iii) designing a derivative of the first ligand identified        in (ii) to provide a SME having a first functional group        reactive with the nucleophile or electrophile on the TM and a        second functional group reactive with a second ligand having        affinity for the second site of interest.

In one embodiment of this aspect of the invention, the SME of step (iii)above is designed such that it is capable of forming an irreversiblecovalent bond with the nucleophile or electrophile of the TM. In apreferred embodiment, the reactive group on the TM is a nucleophile,preferably a thiol, protected thiol, reversible disulfide, hydroxyl,protected hydroxyl, amino, protected amino, carboxyl, or protectedcarboxyl group, and preferred first functional groups on the SME aregroups capable of undergoing SN2-like additions or forming Michael-typeadducts with the nucleophile. SME's designed in this manner are thencontacted with the TM to form an irreversile TM-SME complex. Thiscomplex is then contacted with a plurality of second small organicligand candidates, where such candidates have a functional groupreactive with the SME in the TM-SME complex. As a result, a candidatethat has affinity for the second site of interest on the TM forms areversible covalent bond with the TM-SME complex, whereby a ligandhaving intrinsic binding affinity for the second site of interest isidentified.

In an alternative embodiment of the invention, the SME of step (iii)above is designed to contain a first functional group that forms a firstreversible covalent bond with the nucleophile or electrophile on the TM.The reactive group on the TM preferably is a nucleophile. The reversiblecovalent bond preferably is a disulfide bond which is formed with athiol, protected thiol, or reversible disulfide bond on the TM. SME'sdesigned in this manner are then contacted with the TM either prior toor simultaneously with contacting the TM with a plurality of secondsmall organic ligand candidates, each small organic ligand candidatehaving a free thiol, protected thiol, or a reversible disulfide group,under conditions of thiol exchange, wherein a ligand candidate havingaffinity for the second site of interest on the TM forms a disulfidebond with the TM-SME complex, whereby a second ligand is identified. Theprocess may be performed in the presence of a disulfide reducing agent,such as mercaptoethanol, dithiothreitol (DTT), dithioerythreitol (DTE),mercaptopropanoic acid, glutathione, cysteamine, cysteine,tri(carboxyethyl)phosphine (TCEP), and tris(cyanoethyl)phosphine.

In a particular embodiment, the SME is designed based on selection of asmall organic molecule having a thiol or protected thiol (disulfidemonophore) from a library of such molecules by a Target BiologicalMolecule (TBM) having a thiol at or near a site of interest. In thiscase, the method of this invention is a process comprising:

-   -   (i) contacting a TBM containing or modified to contain a thiol,        protected thiol or reversible disulfide group at or near a first        site of interest on the TBM with a library of small organic        molecules, each small organic molecule having a free thiol or a        reversible disulfide group (disulfide monophores), under        conditions of thiol exchange wherein a library member having        affinity for a first site of interest forms a disulfide bond        with the TBM;    -   (ii) identifying the library member (selected disulfide        monophore) from (i); and    -   (iii) designing a derivative of the library member in (ii) that        is the SME having a first functional group reactive with the        thiol on the TBM and having a second functional group which is a        thiol, protected thiol or reversible disulfide group.

Just as before, the SME can be designed to contain a first functionalgroup that forms an irreversible or reversible covalent bond with theTBM, and can be used to screen small molecule ligand candidates, inparticular libraries of small molecules, as described above, to identifya second ligand.

Thus, in one embodiment, the, SME of step (iii) is designed to contain afirst functional group that forms an irreversible covalent bond with thethiol on the TBM. Preferred first functional groups of this embodimentare groups capable of undergoing SN2 like additions or formingMichael-type adducts with the thiol. SME's designed in this manner arethen contacted with the TBM to form an irreversible TBM-SME complex.This complex is then contacted with second library of small organicmolecules, each small organic molecule having a free thiol or areversible disulfide group, under conditions of thiol exchange whereinthe library member having affinity, preferably the highest affinity, fora second site of interest on the TBM (second ligand) forms a disulfidebond with the TBM-SME complex.

In an alternative embodiment, the small molecule extender (SME) of step(iii) is designed to contain a first functional group that forms a firstreversible disulfide bond with the thiol on the TBM. SME's designed inthis manner are then contacted with the TBM either prior to orsimultaneously with contacting the TBM with a second library of smallorganic molecules, each small organic molecule having a free thiol or areversible disulfide group, under conditions of thiol exchange underconditions wherein the member of the second library having affinity,preferably the highest affinity, for a second site of interest on theTBM (second ligand) forms a disulfide bond with the TBM-SME complex.

The process may be performed in the presence of a disulfide reducingagent, such as those listed above.

Determining the affinity of a ligand candidate (library member) for afirst or second site of interest on a TM or TBM can be carried out bycompetition between different library members in a pool, or bycomparison (i.e. titration) with a reducing agent, such as those listedabove.

In a particular embodiment, the invention concerns a process comprising:

-   -   (i) contacting a Target Biological Molecule (TBM) containing or        modified to contain a nucleophile at or near a site of interest        on the TBM with a small molecule extender having a first        functional group reactive with the nucleophile and having a        second functional group which is a thiol, protected thiol or        reversible disulfide group, thereby forming a TBM-Small Molecule        Extender (TBM-SME) complex;    -   (ii) contacting the TBM-SME complex with a library of small        organic molecules, each small organic molecule (ligand) having a        free thiol, protected thiol or a reversible disulfide group,        under conditions of thiol exchange wherein a library member        having affinity for the site of interest forms a disulfide bond        with the TBM-SME complex thereby forming a TBM-SME-ligand        complex and    -   (iii) determining the ligand from (ii).

In another particular embodiment, the invention concerns a processcomprising:

-   -   (i) providing a Target Biological Molecule (TBM) containing or        modified to contain a reactive nucleophile near a first site of        interest on the TBM;    -   (ii) contacting the TBM from (i) with a small molecule extender        having a group reactive with the nucleophile on the TBM and        having a free thiol or protected thiol;    -   (iii) adjusting the conditions to cause a covalent bond to be        formed between the nucleophile on the TBM and the group on the        small molecule extender thereby forming a covalent complex        comprising the TBM and the small molecule extender, the complex        displaying a free thiol or protected thiol near a second site of        interest on the TBM;    -   (iv) contacting the complex from (iii) with a library of small        organic molecules, each molecule having a free thiol or        exchangeable disulfide linking group, under conditions of thiol        exchange wherein the library member having the highest affinity        for the second site of interest on the TBM forms a disulfide        bond with the complex; and    -   (v) identifying the library member from (iv).

In a particular embodiment, the processes of the present invention maybe performed with a library in which each member forms a disulfide bond.An example of such a library is one in which each member forms acysteamine disulfide. When library members form disulfides, a preferredmolar ratio of reducing agent to total disulfides is from about 1:100 toabout 100:1 and more preferably from about 1:1 to about 50:1.

The tethering process may be performed by contacting members of thedisulfide library one at a time with the TBM or in pools of 2 or more.When pools are used it is preferred to use from 5-15 library members perpool.

In all embodiments, the identity of the small molecules that bind to theSME and/or a site of interest on a TM or TBM may be determined, forexample, by mass spectrometry (MS), or by means of a detectable tag.When mass spectrometry is used to detect the library member that bindsto a TBM and pools are used, it is preferred that each member of thepool differs in molecular weight, preferably by about 10 Daltons.Identification can be performed by measuring the mass of the TBM-librarymember complex, or by releasing the library member form the complexfirst or by using a functional assay, e.g. ELISA, enzyme assay etc.

In a different aspect, the invention concerns a molecule comprising afirst and/or second ligand identified by any of the methods discussedabove. In a particular embodiment, the molecule comprises a first and asecond ligand covalently linked to one another. The covalent linkage maybe provided by any covalent bond, including but not limited to disulfidebonds.

In a further aspect, the invention concerns methods for synthesizingsuch molecules. The molecules obtained can, of course, be furthermodified, for example to impart improved properties, such as solubility,bioavailability, affinity, and half-life. For example, the disulfidebond can be replaced by a linker having greater stability under standardbiological conditions. Possible linkers include, without limitation,alkanes, alkenes, aromatics, heteroaromatics, ethers, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the basic tethering approach forside-directed ligand discovery. A target molecule, containing ormodified to contain a free thiol group (such as a cysteine-containingprotein) is equilibrated with a disulfide-containing library in thepresence of a reducing agent, such as 2-mercaptoethanol. Most of thelibrary members will have little or no intrinsic affinity for the targetmolecule, and thus by mass action the equilibrium will lie toward theunmodified target molecule. However, if a library member does showintrinsic affinity for the target molecule, the equilibrium will shifttoward the modified target molecule, having attached to it the librarymember with a disulfide tether.

FIG. 2 is a schematic illustration of the static extended tetheringapproach. In the first step, a target molecule containing or modified tocontain a free thiol group (such as a cysteine-containing protein) ismodified by a thiol-containing extender, comprising a reactive groupcapable of forming an irreversible covalent bond with the thiol group onthe target molecule, a portion having intrinsic affinity for the targetmolecule, and a thiol group. The complex formed between the targetmolecule and the thiol-containing extender is then used to screen alibrary of disulfide-containing monophores to identify a library memberthat has the highest intrinsic binding affinity for a second bindingsite on the target molecule. LG=ligand; PG=protecting group; R=reactivegroup.

FIG. 3 illustrates the dynamic extended tethering strategy, where theextender is bifunctional and contains two functional groups (usuallydisulfide), each capable of forming reversible covalent bonds.R=reactive group.

FIG. 4 illustrates the chemical synthesis of a specific extender(2,6-dichloro-benzoic acid3-(2-acetylsulfanyl-acetylamino)-4-carboxy-2-oxo-butyl ester), asdescribed in Example 2.

FIG. 5 shows the structural comparison between a known tetrapeptideinhibitor of Caspase-3 and a generic extender synthesized based on theinhibitor.

FIG. 6 shows mass spectra of two representative extended tetheringexperiments.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanismsand Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provideone skilled in the art with a general guide to many of the terms used inthe present application.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

The terms “target,” “Target Molecule,” and “TM” are used interchangeablyand in the broadest sense, and refer to a chemical or biological entityfor which a ligand has intrinsic binding affinity. The target can be amolecule, a portion of a molecule, or an aggregate of molecules. Thetarget is capable of reversible attachment to a ligand via a reversibleor irreversible covalent bond (tether). Specific examples of targetmolecules include polypeptides or proteins (e.g., enzymes, includingproteases, e.g. cysteine, serine, and aspartyl proteases), receptors,transcription factors, ligands for receptors, growth factors, cytokines,immunoglobulins, nuclear proteins, signal transduction components (e.g.,kinases, phosphatases), allosteric enzyme regulators, and the like,polynucleotides, peptides, carbohydrates, glycoproteins, glycolipids,and other macromolecules, such as nucleic acid-protein complexes,chromatin or ribosomes, lipid bilayer-containing structures, such asmembranes, or structures derived from membranes, such as vesicles. Thedefinition specifically includes Target Biological Molecules (TBMs) asdefined below.

A “Target Biological Molecule” or “TBM” as used herein refers to asingle biological molecule or a plurality of biological moleculescapable of forming a biologically relevant complex with one another forwhich a small molecule agonist or antagonist would have therapeuticimportance. In a preferred embodiment, the TBM is a polypeptide thatcomprises two or more amino acids, and which possesses or is capable ofbeing modified to possess a reactive group for binding to members of alibrary of small organic molecules.

The term “polynucleotide”, when used in singular or plural, generallyrefers to any polyribonucleotide or polydeoxribonucleotide, which may beunmodified RNA or DNA or modified RNA or DNA. Thus, for instance,polynucleotides as defined herein include, without limitation, single-and double-stranded DNA, DNA including single- and double-strandedregions, single- and double-stranded RNA, and RNA including single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or includesingle- and double-stranded regions. In addition, the term“polynucleotide” as used herein refers to triple-stranded regionscomprising RNA or DNA or both RNA and DNA. The strands in such regionsmay be from the same molecule or from different molecules. The regionsmay include all of one or more of the molecules, but more typicallyinvolve only a region of some of the molecules. One of the molecules ofa triple-helical region often is an oligonucleotide. The term“polynucleotide” specifically includes DNAs and RNAs that contain one ormore modified bases. Thus, DNAs or RNAs with backbones modified forstability or for other reasons are “polynucleotides” as that term isintended herein. Moreover, DNAs or RNAs comprising unusual bases, suchas inosine, or modified bases, such as tritylated bases, are includedwithin the term “polynucleotides” as defined herein. In general, theterm “polynucleotide” embraces all chemically, enzymatically and/ormetabolically modified forms of unmodified polynucleotides, as well asthe chemical forms of DNA and RNA characteristic of viruses and cells,including simple and complex cells.

A “ligand” as defined herein is an entity which has an intrinsic bindingaffinity for the target. The ligand can be a molecule, or a portion of amolecule which binds the target. The ligands are typically small organicmolecules which have an intrinsic binding affinity for the targetmolecule, but may also be other sequence-specific binding molecules,such as peptides (D-, L- or a mixture of D- and L-), peptidomimetics,complex carbohydrates or other oligomers of individual units or monomerswhich bind specifically to the target. The term “monophore” is usedherein interchangeably with the term “ligand” and refers to a monomericunit of a ligand. The term “diaphore” denotes two monophores covalentlylinked to form a unit that has a higher affinity for the target becauseof the two constituent monophore units or ligands binding to twoseparate but nearby sites on the target. The binding affinity of adiaphore that is higher than the product of the affinities of theindividual components is referred to as “avidity.” The term diaphore isused irrespective of whether the unit is covalently bound to the targetor existing separately after its release from the target. The term alsoincludes various derivatives and modifications that are introduced inorder to enhance binding to the target.

A “site of interest” on a target as used herein is a site to which aspecific ligand binds, which may include a specific sequence ofmonomeric subunits, e.g. amino acid residues, or nucleotides, and mayhave a three-dimensional structure. Typically, the molecularinteractions between the ligand and the site of interest on the targetare non-covalent, and include hydrogen bonds, van der Waals interactionsand electrostatic interactions. In the case of polypeptide, e.g. proteintargets, the site of interest broadly includes the amino acid residuesinvolved in binding of the target to a molecule with which it forms anatural complex in vivo or in vitro.

“Small molecules” are usually less than 10 kDa molecular weight, andinclude but are not limited to synthetic organic or inorganic compounds,peptides, (poly)nucleotides, (oligo)saccharides and the like. Smallmolecules specifically include small non-polymeric (e.g. not peptide orpolypeptide) organic and inorganic molecules. Many pharmaceuticalcompanies have extensive libraries of such molecules, which can beconveniently screened by using the extended tethering approach of thepresent invention. Preferred small molecules have molecular weights ofless than about 300 DA and more preferably less than about 650 Da.

The term “tether” as used herein refers to a structure which includes amoiety capable of forming a reversible or reversible covalent bond witha target (including Target Biological Molecules as hereinabove defined),near a site of interest.

The phrase “Small Molecule Extender” (SME) as used herein refers to asmall organic molecule having a molecular weight of from about 75 toabout 1,500 daltons and having a first functional group reactive with anucleophile or electrophile on a TM and a second functional groupreactive with a ligand candidate or members of a library of ligandcandidates. Preferably, the first functional group is reactive with anucleophile on a TBM (capable of forming an irreversible or reversiblecovalent bond with such nucleophile), and the reactive group at theother end of the SME is a free or protected thiol or a group that is aprecursor of a free of protected thiol. In one embodiment, at least aportion of the small molecule extender is capable of forming anoncovalent bond with a first site of interest on the TBM (i.e. has aninherent affinity for such first site of interest). Included within thisdefinition are small organic (including non-polymeric) moleculescontaining metals such as Cd, Hg and As which may form a bond with thenucleophile. e.g. SH of the TBM.

The phrase “reversible covalent bond” as used herein refers to acovalent bond which can be broken, preferably under conditions that donot denature the target. Examples include, without limitation,disulfides, Schiff-bases, thioesters, and the like.

The term “reactive group” with reference to a ligand is used to describea chemical group or moiety providing a site at which covalent bond withthe ligand candidates (e.g. members of a library or small organiccompounds) may be formed. Thus, the reactive group is chosen such thatit is capable of forming a covalent bond with members of the libraryagainst which it is screened.

The term “antagonist” is used in the broadest sense and includes anyligand that partially or fully blocks, inhibits or neutralizes abiological activity exhibited by a target, such as a TBM. In a similarmanner, the term “agonist” is used in the broadest sense and includesany ligand that mimics a biological activity exhibited by a target, suchas a TBM, for example, by specifically changing the function orexpression of such TBM, or the efficiency of signaling through such TBM,thereby altering (increasing or inhibiting) an already existingbiological activity or triggering a new biological activity.

The phrases “modified to contain” and “modified to possess” are usedinterchangeably, and refer to making a mutant, variant or derivative ofthe target, or the reactive nucleophile or electrophile, including butnot limited to chemical modifications. For example, in a protein one cansubstitute an amino acid residue having a side chain containing anucleophile or electrophile for a wild-type residue. Another example isthe conversion of the thiol group of a cysteine residue to an aminegroup.

The term “reactive nucleophile” as used herein refers to a nucleophilethat is capable of forming a covalent bond with a compatible functionalgroup on another molecule under conditions that do not denature ordamage the target, e.g. TBM. The most relevant nucleophiles are thiols,alcohols, activated carbonyls, epoxides, aziridines, aromaticsulfonates, hemiacetals, and amines. Similarly, the term “reactiveelectrophile” as used herein refers to an electrophile that is capableof forming a covalent bond with a compatible functional group on anothermolecule, preferably under conditions that do not denature or otherwisedamage the target, e.g. TMB. The most relevant electrophiles are imines,carbonyls, epoxides, aziridies, sulfonates, and hemiacetals.

A “first site of interest” on a target, e.g. TBM refers to a site thatcan be contacted by at least a portion of the SME when it is covalentlybound to the reactive nucleophile or electrophile. The first site ofinterest may, but does not have to possess the ability to form anoncovalent bond with the SME.

The phrases “group reactive with the nucleophile,” “nucleophile reactivegroup,” “group reactive with an electrophile,” and “electrophilereactive group,” as used herein, refer to a functional group on the SMEthat can form a covalent bond with the nucleophile/electrophile on theTM, e.g. TBM under conditions that do not denature or otherwise damagethe TM, e.g. TBM.

The term “protected thiol” as used herein refers to a thiol that hasbeen reacted with a group or molecule to form a covalent bond thatrenders it less reactive and which may be deprotected to regenerate afree thiol.

The phrase “adjusting the conditions” as used herein refers tosubjecting a target, such as a TBM to any individual, combination orseries of reaction conditions or reagents necessary to cause a covalentbond to form between the ligand and the target, such as a nucleophileand the group reactive with the nucleophile on the SME, or to break acovalent bond already formed.

The term “covalent complex” as used herein refers to the combination ofthe SME and the TM, e.g. TBM which is both covalently bonded through thenucleophile/electrophile on the TM, e.g. TBM with the group reactivewith the nucleophile/electrophile on the SME, and non-covalently bondedthrough a portion of the small molecule extender and the first site ofinterest on the TM, e.g. TBM.

The phrase “exchangeable disulfide linking group” as used herein refersto the library of molecules screened with the covalent complexdisplaying the thiol-containing small molecule extender, where eachmember of the library contains a disulfide group that can react with thethiol or protected thiol displayed on the covalent complex to form a newdisulfide bond when the reaction conditions are adjusted to favor suchthiol exchange.

The phase “highest affinity for the second site of interest” as usedherein refers to the molecule having the greater thermodynamic stabilitytoward the second site of interest on the TM, e.g. TBM that ispreferentially selected from the library of disulfide-containing librarymembers.

“Functional variants” of a molecule herein are variants having anactivity in common with the reference molecule.

“Active” or “activity” means a qualitative biological and/orimmunological property.

2. Targets

Targets, such as target biological molecules (TBMs), that find use inthe present invention include, without limitation, molecules, portionsof molecules and aggregates of molecules to which a ligand candidate maybind, such as polypeptides or proteins (e.g., enzymes, receptors,transcription factors, ligands for receptors, growth factors,immunoglobulins, nuclear proteins, signal transduction components,allosteric enzyme regulators, and the like), polynucleotides, peptides,carbohydrates, glycoproteins, glycolipids, and other macromolecules,such as nucleic acid-protein complexes, chromatin or ribosomes, lipidbilayer-containing structures, such as membranes, or structures derivedfrom membranes, such as vesicles. The target can be obtained in avariety of ways, including isolation and purification from naturalsource, chemical synthesis, recombinant production and any combinationof these and similar methods.

Preferred enzyme target families are cysteine proteases, aspartylproteases, serine proteases, metalloproteases, kinases, phosphatases,polymerases and integrases. Preferred protein:protein targets are4-helical cytokines, trimeric cytokines, signaling modules,transcription factors and chemokines.

In a particularly preferred embodiment, the target is a TBM, and evenmore preferably is a polypeptide, especially a protein. Polypeptides,including proteins, that find use herein as targets for binding ligands,preferably small organic molecule ligands, include virtually anypolypeptide (including short polypeptides also referred to as peptides)or protein that comprises two or more binding sites of interest, andwhich possesses or is capable of being modified to possess a reactivegroup for binding to a small organic molecule or other ligand (e.g.peptide). Polypeptides of interest may be obtained commercially,recombinantly, by chemical synthesis, by purification from naturalsource, or otherwise and, for the most parts are proteins, particularlyproteins associated with a specific human disease or condition, such ascell surface and soluble receptor proteins, such as lymphocyte cellsurface receptors, enzymes, such as proteases (e.g., serine, cysteine,and aspartyl proteases) and thymidylate synthetase, steroid receptors,nuclear proteins, allosteric enzymes, clotting factors, kinases (bothserine and threonine) and dephosphorylases (or phophatases, eitherserine/threonine or protein tyrosine phosphatases, e.g. PTP's,especially PTP1B), bacterial enzymes, fungal enzymes and viral enzymes(especially those associate with HIV, influenza, rhinovirus and RSV),signal transduction molecules, transcription factors, proteins orenzymes associated with DNA and/or RNA synthesis or degradation,immunoglobulins, hormones, receptors for various cytokines including,for example, erythropoietin (EPO), granulocyte colony stimulating(G-CSF) receptor, granulocyte macrophage colony stimulating (GM-CSF)receptor, thrombopoietin (TPO), interleukins, e.g. IL-2, IL-3, IL-4,IL-5, IL-6, IL-10, IL-11, IL-12, growth hormone, prolactin, humanplacental lactogen (LPL), CNTF, oncostatin, various chemokines and theirreceptors, such as RANTES MIPβ, IL-8, various ligands and receptors fortyrosine kinase, such as insulin, insulin-like growth factor 1 (IGF-1),epidermal growth factor (EGF), heregulin-α and heregulin-β, vascularendothelial growth factor (VEGF), placental growth factor (PLGF), tissuegrowth factors (TGF-α and TGF-β), nerve growth factor (NGF), variousneurotrophins and their ligands, other hormones and receptors such as,bone morphogenic factors, follicle stimulating hormone (FSH), andluteinizing hormone (LH), trimeric hormones including tissue necrosisfactor (TNF) and CD40 ligand, apoptosis factor-1 and -2 (AP-1 and AP-2),p53, bax/bcl2, mdm2, caspases, and proteins and receptors that share 20%or more sequence identity to these.

An important group of human inflammation and immunology targetsincludes: IgE/IgER, ZAP-70, lck, syk, ITK/BTK, TACE, Cathepsin S and F,CD11a, LFA/ICAM, VLA-4, CD28/B7, CTLA4, TNF alpha and beta, (and the p55and p75 TNF receptors), CD40L, p38 map kinase, IL-2, IL-4, Il-13, IL-15,Rac 2, PKC theta, IL-8, TAK-1, jnk, IKK2 and IL-18.

Still other important specific targets include: caspases 1, 3, 8 and 9,IL-1/IL-1 receptor, BACE, HIV integrase, PDE IV, Hepatitis C helicase,Hepatitis C protease, rhinovirus protease, tryptase, cPLA (cytosolicPhospholipase A2), CDK4, c-jun kinase, adaptors such as Grb2, GSK-3,AKT, MEKK-1, PAK-1, raf, TRAF's 1-6, Tie2, ErbB 1 and 2, FGF, PDGF,PARP, CD2, C5a receptor, CD4, CD26, CD3, TGF-alpha, NF-kB, IKK beta,STAT 6, Neurokinnin-1, PTP-1B, CD45,Cdc25A, SHIP-2, TC-PTP, PTP-alpha,LAR and human p53, bax/bcl2 and mdm2.

The target, e.g. a TBM of interest will be chosen such that it possessesor is modified to possess a reactive group which is capable of forming areversible or irreversible covalent bond with a ligand having intrinsicaffinity for a site of interest on the target. For example, many targetsnaturally possess reactive groups (for example, amine, thiol, aldehyde,ketone, hydroxyl groups, and the like) to which ligands, such as membersof an organic small molecule library, may covalently bond. For example,polypeptides often have amino acids with chemically reactive side chains(e.g., cysteine, lysine, arginine, and the like). Additionally,synthetic technology presently allows the synthesis of biological targetmolecules using, for example, automates peptide or nucleic acidsynthesizers, which possess chemically reactive groups at predeterminedsites of interest. As such, a chemically reactive group may besynthetically introduced into the target, e.g. a TBM, during automatedsynthesis.

In one particular embodiment, the target comprises at least a firstreactive group which, if the target is a polypeptide, may or may not beassociated with a cysteine residue of that polypeptide, and preferablyis associated with a cysteine residue of the polypeptide, if the tetherchosen is a free or protected thiol group (see below). The targetpreferably contains, or is modified to contain, only a limited number offree or protected thiol groups, preferably not more than about 5 thiolgroups, more preferably not more than about 2 thiol groups, morepreferably not more than one free thiol group, although polypeptideshaving more free thiol groups will also find use. The target, such asTBM, of interest may be initially obtained or selected such that italready possesses the desired number of thiol groups, or may be modifiedto possess the desired number of thiol groups.

When the target is a polynucleotide, a tether can, for example, beattached to the polynucleotide on a base at any exocyclic amine or anyvinyl carbon, such as the 5- or 6-position of pyrimidines, 8- or2-positions of purines, at the 5′ or 3′ carbons, at the sugar phosphatebackbone, or at internucleotide phosphorus atoms. However, a tether canbe introduced also at other positions, such as the 5-position ofthymidine or uracil. In the case of a double-stranded DNA, for example,a tether can be located in a major or minor groove, close to the site ofinterest, but not so close as to result in steric hindrance, which mightinterfere with binding of the ligand to the target at the site ofinterest.

Those skilled in the art are well aware of various recombinant,chemical, synthesis and/or other techniques that can be routinelyemployed to modify a target, e.g. a polypeptide of interest such that itpossesses a desired number of free thiol groups that are available forcovalent binding to a ligand candidate comprising a free thiol group.Such techniques include, for example, site-directed mutagenesis of thenucleic acid sequence encoding the target polypeptide such that itencodes a polypeptide with a different number of cysteine residues.Particularly preferred is site-directed mutagenesis using polymerasechain reaction (PCR) amplification (see, for example, U.S. Pat. No.4,683,195 issued 28 Jul. 1987; and Current Protocols In MolecularBiology, Chapter 15 (Ausubel et al., ed., 1991). Other site-directedmutagenesis techniques are also well known in the art and are described,for example, in the following publications: Ausubel et al, supra,Chapter 8; Molecular Cloning: A Laboratory Manual., 2^(nd) edition(Sambrook et al., 1989); Zoller et al., Methods Enzymol. 100:468-500(1983); Zoller & Smith, DNA 3:479-488 (1984); Zoller et al., Nucl. AcidsRes., 10:6487 (1987); Brake et al., Proc. Natl. Acad. Sci. USA81:4642-4646 (1984); Botstein et al, Science 229:1193 (1985); Kunkel etal, Methods Enzymol. 154:367-82 (1987), Adelman et al, DNA 2:183 (1983);and Carter et al., Nucl. Acids Res., 13:4331 (1986). Cassettemutagenesis (Wells et al., Gene, 34:315 [1985]), and restrictionselection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA,317:415 [1986]) may also be used.

Amino acid sequence variants with more than one amino acid substitutionmay be generated in one of several ways. If the amino acids are locatedclose together in the polypeptide chain, they may be mutatedsimultaneously, using one oligonucleotide that codes for all of thedesired amino acid substitutions. If, however, the amino acids arelocated some distance from one another (e.g. separated by more than tenamino acids), it is more difficult to generate a single oligonucleotidethat encodes all of the desired changes. Instead, one of two alternativemethods may be employed. In the first method, a separate oligonucleotideis generated for each amino acid to be substituted. The oligonucleotidesare then annealed to the single-stranded template DNA simultaneously,and the second strand of DNA that is synthesized from the template willencode all of the desired amino acid substitutions. The alternativemethod involves two or more rounds of mutagenesis to produce the desiredmutant.

Sources of new reactive groups, e.g. cysteines can be placed anywherewithin the target. For example, if a cysteine is introduced onto thesurface of the protein in an area known to be important forprotein-protein interactions, small molecules can be selected that bindto and block this surface.

The following tables exemplify target biological molecules (TBM's) thatcan be used in accordance with the present invention. ImmunologyIndications IL-6 Inflammation B7/CD28 Graft rejection CD4Immunosuppression CD3 Immunosuppression CD2 Renal Transplantation c-mafInflammation/Immunosuppression CD11a/LFA1 (ICAM)Immunosuppression/Inflammation

Enzymes Indications Phospholipase A2 Inflammation ZAP-70Immunosuppression Phophodiesterase IV Asthma Interleukin convertingenzyme (ICE) Inflammation Inosine monophosphate dehydrogenase Autoimmunediseases Tryptase Psoriasis/asthma CDK4 Cancer mTOR ImmunosuppressionPARP (Cell death pathway) Stroke Phosphatases Cancer Raf Cancer JNK3Neurodegeneration MEK Cancer GSK-3 Diabetes FABI (Fatty acidbiosynthesis) Bacterial FABH (Fatty acid biosynthesis) Bacterial BACEAlzheimer's IkB-ubiquitin Ligase Inflammation/diabetes Lysophosphatidicacid acetlytransferase CD26 (dipeptidyl peptidase IV) Akt TNF convertingenzyme Inflammation

Viral Targets Indications Rhinovirus protease Common cold Parainfluenzaneuraminidase Colds/Veterinary uses HIV fusion gp41 HIVinfection/treatment Hepatitis C Helicase Hepatitis Hepatitis C proteaseHepatitis

Protein-Protein Targets Indications ErbB Receptors Cancer Neurokinin-1Inflammation, Migraine IL-9 Asthma FGF Angiogenesis PDGF AngiogenesisTIE2 Angiogenesis NFκB Dimerization Inflammation Tissue Factor/FactorVII Cardiovascular Disease Selectins Inflammation TGF-α AngiogenesisAngiopoietin I Angiogenesis APAF-1/Caspase 9 CARD Stroke Bcl-2 Cancer

7-Transmembrane Indications IL-8 Stroke, inflammation RantesInflammation, Migraine CC Chemokine Receptors Asthma GPR 14/UrotensinAngiogenesis Orexin/Receptor Appetite C5a receptor Sepsis/crohn'sdisease Histamine H3 receptor Allergy CCR5 HIV attachment

Target PDB Codes Accession No. Crystal Structure Ref. BACE 1FKN GBAAF13715 Hong, L. et al., Science. 290(5489): 150-3 (2000). Caspase 11BMQ SWS P29466 Okamoto, Y., et al., Chem Pharm Bull (Tokyo), 47(1):11-21 (1999). Caspase 4 none SWS P49662 NA Caspase 5 none SWS P51878 NACaspase 3 1CP3 SWS P42574 Mittl, PR, et al., J Biol Chem, 272(10):6539-47(1997). Caspase 8 1I4E, 1QTN SWS P08160; Xu, G., et al., Nature,410(6827): 494-7 GB BAB32555 (2001). Caspase 9 3YGS SWS P55211 Qin, H.,et al., Nature, 399(6736): 549-57 (1999). RHV·Prot 1CQQ SWS P04936Matthews, D., et al., 96(20): 11000-7 (1999). Cathepsin K 1MEM SWSP43235 McGrath, ME, et al., Nat Struct Biol, 4(2): 105-(1997). CathepsinS 1BXF SWS P25774 Fengler, A., et al., Protein (model) Eng, 11(11):1007-13(1998). Tryptase 1A0L SWS P20231 Pereira, P. J. et al., Nature,392(6673): 306-11 (1998). HCV Prot 1A1R, SWS Q81755 Di Marco, et al., JBiol Chem. IDY9 275(10): 7152-7(2000). CD26 none SWS P27487 NA TACE 1BKCGB U69612 Maskos, K., et al., PNAS, 95(7): 3408-12 (1998). ZAP-70 noneSWS P43403 NA p38 MAP 1P38 SWS P47811 Wang, Z., et al., PNAS, 94(6):2327-32 (1997). CDK-4 none SWS Q9XTB6 NA c-jun kinase NA SWS P45983 (C-NA Jun Kinase-1) NA SWS P45984 (C- NA Jun Kinase-2) 1JNK SWS P53779 (C-NA Jun Kinase-3) GSK-3 NA SWS P49840 NA (GSK-3A) NA SWS P49841 NA(GSK-3B) AKT none SWS P31749 NA MEK none SWS Q02750 NA Raf none SWSP04049 NA TIE-2 none SWS Q02763 NA ILK none SWS Q13418 NA IkB NA SWSO15111 NA (IKappaBKinase) NA SWS O14920 NA (IKappBKinBeta) Jak1 none SWSP23458 NA Jak2 none SWS O60674 NA Jak3 none SWS P52333 NA Tyk2 none SWSP29597 NA EGF Kinase see Vasc. Endo. Growth Factor Receptor (VEGFR) andEGFR both with tyrosine kinase activity(Below): VEGFR2/KD NA SWS P35968NA R Kinase EGFR NA SWS P00533 NA TC-PTP NA SWS P17706 NA: T-cellProtein Tyrosine Phosphatase CDC25A NA SWS P30304 NA CDC25A NA GB O14757NA CDK (CHK1) CD45 NA SWS P08575 NA PTP alpha NA SWS P18433 NA pol IIINA SWS O14802 NA; DNA directed RNA polymerase III (PolRIIIA) mur-DLigase NA GB O14802 (E. Coli) NA NA SWS P14900 (E. Coli) NA SHP NA SWSQ15466 NA PTP-1B 1PTP SWS P00760 Finer-Moore, JS, et al., Proteins,12(3): 203-22(1992). SHIP-2 none SWS Q9R1V2 NA MEKK-1 NA SWS Q13233 NAPAK-1 NA SWS Q13153 NA ICAM-1 NA SWS P05362 Bella, J., et al., Proc NatlAcad Sci USA, 95(8): 4140-5 (1998). CD11A/LFA-1 NA SWS P20701 Qu, A., etal., Proc Natl Acad Sci USA, 92(22: 10277-81 (1995) TAF1 UNSURE UNSURE(see UNSURE below) NA SWS Q99142 (?? NA; tobacco Tumor Activating FactorTobacco Prot.) NA GB AAB30018 NA; Tumor-derived Adhesion Factor NA GBD45198 NA; Template Activating Factor HIV-Integrase 1BL3 (2.0) SWSP12497 Maignan, S., et al., J Mol Biol, 282(2): 359-68 (1998). 1EXQ SWSP04585 Chen, J. C-H., et al., PNAS USA, 97(15): 8233-8 (2000) NA SWSO56380 NA 1HYZ SWS O56381; Molteni, V., et al., Acta Crystallogr GBAAC37875 D Bio Crystallog., 57: 536-44 (2001). 1HYV GB AAC37875 Molteni,V., et al., Acta Crystallogr D Biol Crystallogr., 57(Pt 4): 536-44(2001). NA SWS O56382 NA NA SWS O56383 NA NA SWS O56384 NA NA SWS 056385NA HCV-Helicase 1N13, SWS Q81755 Di Marco, S., et al., J Biol Chem.,1DY9, (1DY9) 275(10): 7152-7 (2000). others (Integrase) 1HEI SWS P2664Yao, N., et al., Nat Struct Biol, 4(6): 463-7 (Helicase) (1997). Infl.1A4G; many SWS P27907 Taylor, N., et al., J Med Chem, Neuraminidase41(6): 798-807 (1998). PDE-IV 1FOJ SWS Q07343 Xu, R. X., et al.,Science., (PDE4B2B) 288(5472): 1822-5 (2000). cPLA-2 1CJY SWS P47712Dessen, A., et al., Cell., 97(3): 349-60 (1999). IL-2 NA (in- SWS P01585NA house IL-4 1HIK(apo) SWS P05112 Muller, T., et al., J Mol Biol,247(2): 360-72 (2.60) (1995). 1IAR SWS P05112 Hage, T., et al., Cell.,97(2): 271-81 (complex) (2.30)** (1999). IL-4R 1IAR SWS P24394 Hage, T.,et al., Cell., 97(2): 271-81 (1999). IL-5 1HUL SWS P05113 Milburn, M.V., et al., Nature, 363(6425): 172-6 (1993). IL-6 1I1R(viral GB AAB62676Chow, D., et al., Science, IL6) (2.6) 291(5511): 2150-5 (2001). 1ALU SWSP05231 Somers, W., et al., EMBO J, 16(5): 989-97 (1.9) (1997). IL-7 1IL7SWS P13232 Cosenza, L., et al., Protein Sci., 9(5): 916-26 (model)(2000). IL-9 none SWS P15248 NA IL-13 1GA3 SWS P35225 NA (NMR) TNF 1TNFSWS P01375 Eck, MJ, et al., J Biol (TNF-alpha) Chem, 264(29)17595-605(1989). CD-40 L 1ALY SWS P29965 Karpusas, M., et al.,Structure, 3(12): 1426 (1995). OPGL none SWS O14788 NA BAFF none SWSQ9Y275 NA TRAIL 1DG6 (1.30) GB AAC50332 Hymowitz, S. G., et al.,Biochemistry, 39(4): 633-40 (2000). 1DU3 (2.2) SWS P50591; Cha SS, etal., J Biol Chem, GB AAC50332 275(40): 31171-7 (2000). 1D2Q GB AAC50332Cha and Oh, Immunity, 11(2): 253-61 (1999). IL-1 NA SWS P01584 NA (IL-1B Cytokine) IL-1R 1G0Y SWS P14778 Vigers, GPA, et al., J Biol Chem.,275(47): 36927-33 (2000). IL-8 1QE6 SWS P10145 Gerber, N., et al.,Proteins, 38(4): 361-7 (2000). RANTES-R NA SWS P32246 NA RANTES NA GBXP_035842 NA NA SWS P13501 NA; (T-cell specific RANTES protein) MCP-1 NASWS Q14805 NA; (Metaphase chromosomal protein) MCP-1 1D0K SWS P13500Lubowski, J., et al., Nat Struct Biol., 4(1): 64-9 (1997). MCP-3 NA SWSP80098 Nat Struct Biol, 4(1): 64-9 (1997). TRAF-A NA SWS Q13077 NA(TRAF-1?) (TRAF-1) Target PDB Codes Accession No. Crystal Structure Ref.TRAF-B NA SWS Q12933 NA (TRAF-2?) (TRAF-2) 1D00 GB S56163 Ye, H., etal., Mol Cell, 4(3): 321-30 (TRAF-2) (TRAF-2) (2.0) (1999). TRAF-C NASWS Q13114 NA (TRAF-3?) (TRAF-3) TRAF-D NA GB XP_008483 NA (TRAF-4?)(TRAF-4) TRAF-E NA GB XP_010656 NA (TRAF-5?) (TRAF-5) VEGF 1FLT SWSP15692 Wiesmann, C., et al., Cell, 91(5): 695-704 (1997). Mineral NA SWSP08235 NA Corticoid R. Estrogen 3ERD SWS P03372 Shiau, A. K., Barstad,D., Loria, P. M., Receptor Cheng, L., Kushner, P. J., Agard, D. A.,Greene, G. L., Cell, 95(7): 927-37 (1998). Progesterone 1A28 SWS P06401Williams, S. P., Sigler, P. B, Nature, Rec. 393(6683): 392-6 (1998).NF-kappa-B-1 SWS P19838 P53 NA SWS P04637 NA Y1CQ GB AAA59989 Kussie, P.H., et al., (2.3) Science, 74(5289): 948-53 (1996). MDM2 1YCR SWS Q00987Kussie, P. H., et al., Science, 74(5289): 948-53 (1996). STAT6 NA SWSP42226 NA IL4R-alpha NA SWS P24394 NA IL6R-alpha NA SWS P08887 NAIL6R-beta 1BQU SWS P40189 Bravo, J., Staunton, D., Heath, J. K., chainJones, E. Y., EMBO J, 17(6): 1665-74 (1998). IL5R-alpha NA SWS Q01344 NAIL7R NA SWS P16871 NA IL2R-alpha NA SWS P01589 NA IL2R-beta NA SWSP14784 NA HIV GP41 1AIK SWS P19551 Chan, D. C., Fass, D., Berger, J. M.,Kim, P. S., Cell, 89(2): 263-73 (1997). HIV GP41 1AIK SWS P04582 Chan,D. C., Fass, D., Berger, J. M., Kim, P. S., Cell, 89(2): 263-73 (1997).HIV GP41 SWS P03378 HIV GP41 SWS P03375 HIV GP41 SWS P04582 HIV GP41 SWSP12488 HIV GP41 SWS P03377 HIV GP41 SWS P05879 HIV GP41 SWS P04581 HIVGP41 SWS P04578 HIV GP41 SWS P04624 HIV GP41 SWS P12489 HIV GP41 SWSP20871 HIV GP41 SWS P31819 HIV GP41 SWS Q70626 HIV GP41 SWS P04583 HIVGP41 SWS P19551 HIV GP41 SWS P05577 HIV GP41 SWS P18799 HIV GP41 SWSP20888 HIV GP41 SWS P03376 HIV GP41 SWS P04579 HIV GP41 SWS P19550 HIVGP41 SWS P19549 HIV GP41 SWS P05878 HIV GP41 SWS P31872 HIV GP41 SWSP05880 HIV GP41 SWS P35961 HIV GP41 SWS P12487 HIV GP41 SWS P04580 HIVGP41 SWS P05882 HIV GP41 SWS P05881 HIV GP41 SWS P18094 HIV GP41 SWSP24105 HIV GP41 SWS P17755 HIV GP41 SWS P15831 HIV GP41 SWS P18040 HIVGP41 SWS Q74126 HIV GP41 SWS P05883 HIV GP41 SWS P04577 HIV GP41 SWSP32536 HIV GP41 SWS P12449 HIV GP41 SWS P20872 c-mal NA GB NP_071884 NA;T-cell differentiation protein NA GB CAA54102 NA NA GB XP_017128 NA MalNA SWS P21145 NA; T-LYMPHOCYTE MATURATION-ASSOCIATED PROTEIN NA SWSP01732 NA; T-LYMPHOCYTE DIFFERENTIATION ANTIGEN T8/CD8(?) Her-1 NA SWSP34704 NA: Cell Signaling in C. elegans Sex Determination Her-2 NA SWSP04626 NA; RECEPTOR PROTEIN-TYROSINE KINASE ERBB-2 E2F-1 NA SWS Q01094NA E2F-2 NA SWS Q14209 NA E2F-3 NA SWS O00716 NA E2F-4 NA SWS Q16254 NAE2F-5 NA SWS Q15329 NA E2F-6 NA SWS O75461 NA Cyclin A 1QMZ SWS P20248Brown, N. R., et al., Nat Cell Biol., 1(7): 438-43 (1999). mTOR/FRAP1NSG SWS P42345 Liang, J., et al., Acta Crystall D Biol Crystall, 55 (Pt4): 736-44 (1995). Survivin 1F3H SWS O15392 Verdecia, M. A., et al., NatStruct Biol., 7(7): 602-8 (2000). FGF-1 1EV2 SWS P05230 Plotnikov, A.N., et al., Cell., 101(4): 413-24 (2000).(Heparin Binding Growth FactorI) Basic FGF 1FGK SWS P11362 Mohammadi, M., et al., Cell, 86(4): 577-87Rec. I (1996).(Basic FGF Rec. I) FGF-2 1CVS SWS P09038 Plotnikov, A. N.,et al., Cell, 98(5): 641-50 (1999). FGF-3 NA SWS P11487 NA FGF-4 NA SWSP08620 NA FGF-5 NA SWS P12034 NA FGF-6 NA SWS P10767 NA FGF-7 NA SWSP21781 NA FGF-8 NA SWS P55075 NA FGF-9 1IHK SWS P31371 Plotnikov, A. N.,et al., J Biol Chem., 276(6): 4322-9 (2001). PARP NA SWS P09874 NAPDGF-alpha NA SWS P04085 NA PDGF-beta NA SWS P01127 NA C5a receptor NASWS P21730 NA CCR5 NA SWS P51681(CC NA Chemo R-V) GPR14/Urote NA SWSQ9UKP6 NA nsin IIR) Tissue Factor 2HFT SWS P13726 Muller, Y. A., et al.,J Mol Biol, 256(1): 144-59 (1996). Factor VII 1JBU SWS P08709 Eigenbrot,C., et al., Structure, 9: 627 (2001). Histamine H3 NA GB CAC39434 NArec. Neurokinin-1 NA GB SPHUB NA orexin NA SWS O43613 NA receptor-1orexin NA SWW O43614 NA receptor-2 CD-3 delta NA SWS P04234 NA chainCD-3 epsilon NA SWS P07766 NA chain CD-3 gamma NA SWS P09693 NA chainCD-3 zeta NA SWS P20963 NA chain CD-4 1CDJ SWS P01730 Wu, H., et al.,Proc Natl Acad Sci USA, 93(26): 15030-5 (1996). TGF-alpha NA SWS P01135NA TGF-beta-1 NA SWS P01137 NA TGF-beta-2 NA SWS P08112 NA TGF-beta-3 NASWS P10600 NA TGF-beta-4 NA SWS O00292 NA GRB2 1GRI SWS P29354 MaignanS, et al., Science, (3.1) 268(5208): 291-3 (1995). 1ZFP SWS P29354Rahuel, J., et al., J Mol Biol, (1.8) 279(4): 1013-22 (1998). 1BMB SWSP29354 Ettmayer, P., et al., J Med Chem, (1.8) 42(6): 971-80 (1999). LCK1LKK SWS Tong, L., et al., J Mol Biol, 256(3): 601-10 P06239; (2nd =P07100) (1996). SRC 2SRC SWS P12931 Xu, W., et al., Mol Cell., 3(5):629-38 (1999). TRAFs? NA SWS Q13077 NA (TRAF-1) 1CZZ GB S56163 Ye, H.,et al., Mol Cell, 4(3): 321-30 (TRAF-2) (TRAF-2) (2.7) (1999). 1CZY GBS56163 Ye, H., et al., Mol Cell, 4(3): 321-30 (TRAF-2) (TRAF-2) (2.0)(1999). 1D00 GB S56163 Ye, H., et al., Mol Cell, 4(3): 321-30 (TRAF-2)(TRAF-2) (2.0) (1999). NA SWS Q12933 NA (TRAF-2) 1FLK GB Q13114 Ni,C.-Z., et al., Proc Natl Acad Sci USA., (TRAF-3) (TRAF-3) (2.8) 97(19):10395-9 (2000). BAX/BCL-2 NA SWS Q07812 NA (BAX alpha) NA SWS Q07814 NA(BAX beta) NA SWS Q07815 NA (BAX gamma) NA SWS P55269 NA (BAX delta) NASWS P10415 NA (BCL-2) IgE 1F6A (3.5) SWS P01854 Garman, S. C., et al.,T. S., Nature., (IgE chain C) 406(6793): 259-66 (2000). IgER NA SWSP06734 NA (IgE Fc Receptor) 1F6A (3.5) SWS P12319 Garman, S. C., et al.,T. S., Nature., (IgE Fc Rec. 406(6793): 259-66 (2000). alpha) 1F2Q (2.4)SWS P12319 Garman, S. C., Kinet, J. P., Jardetzky, T. S., (IgE Fc Rec.Cell, 95(7): 951-61 (1998). alpha) NA SWS Q01362 NA (IgE FcRec. Beta) NASWS P30273 NA (IgE FcRec. Gama) Rhinovirus NA SWS P03303 NA Protease(HRV-14 polyprot.) NA SWS P12916 NA (HRV-1B) 1CQQ SWS P04936 Matthews,D., et al., Proc Natl Acad Sci (HRV-2) (1.85) USA, 96(20): 11000-7(1999). NA SWS P07210 NA (HRV-89) 1C8M SWS Q82122 Chakravarty, S., etal., to be published (HRV-16) B7/CD28LG/ 1DR9 SWS P33681 Ikemizu, S., etal., Immunity. 2000 CD80 Jan; 12(1): 51-60. CD28 NA SWS P10747 NA APAF1NA SWS O14727 NA3. Site(s) of Interest

Broadly, the “site of interest” on a particular target, such as a TargetBiological Molecule (TBM), is defined by the residues that are involvedin binding of the target to a molecule with which it forms a naturalcomplex in vivo or in vitro. If the target is a peptide, polypeptide, orprotein, the site of interest is defined by the amino acid residues thatparticipate in binding to (usually by non-covalent association) to aligand of the target.

When, for example, the target biological molecule is a protein thatexerts its biological effect through binding to another protein, such aswith hormones, cytokines or other proteins involved in signaling, it mayform a natural complex in vivo with one or more other proteins. In thiscase, the site of interest is defined as the critical contact residuesinvolved in a particular protein:protein binding interface. Criticalcontact residues are defined as those amino acids on protein A that makedirect contact with amino acids on protein B, and when mutated toalanine decrease the binding affinity by at least 10 fold and preferablyat least 20 fold, as measured with a direct binding or competition assay(e.g. ELISA or RIA). See (A Hot Spot of Binding Energy in aHormone-Receptor Interface by Clackston and Wells Science 267:383-386(1995) and Cunningham and Wells J. Mol. Biol, 234:554-563 (1993)). Alsoincluded in the definition of a site of interest are amino acid residuesfrom protein B that are within about 4 angstroms of the critical contactresidues identified in protein A.

Scanning amino acid analysis can be employed to identify one or moreamino acids along a contiguous sequence. Among the preferred scanningamino acids are relatively small, neutral amino acids. Such amino acidsinclude alanine, glycine, serine, and cysteine. Alanine is typically apreferred scanning amino acid among this group because it eliminates theside-chain beyond the beta-carbon and is less likely to alter themain-chain conformation of the variant (Cunningham and Wells, Science,244: 1081-1085 (1989)). Alanine is also typically preferred because itis the most common amino acid. Further, it is frequently found in bothburied and exposed positions (Creighton, The Proteins, (W.H. Freeman &Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). If alaninesubstitution does not yield adequate amounts of variant, an isotericamino acid can be used.

When the target biological molecule is an enzyme, the site of interestcan include amino acids that make contact with, or lie within, about 4angstroms of a bound substrate, inhibitor, activator, cofactor orallosteric modulator of the enzyme. By way of illustration, when theenzyme is a protease, the site of interest would include the substratebinding channel from P4 to P4′, residues involved in catalytic function(e.g. the catalytic triad) and any cofactor (e.g. Zn) binding site. Forprotein kinases, the site of interest would include thesubstrate-binding channel (as above) in addition to the ATP bindingsite. For dehydrogenases, the site of interest would include thesubstrate binding region as well as the site occupied by NAD/NADH. Inhydrolases such as PDE4, the site of interest would include all residuescontacting the cAMP substrate, as well as residues involved in bindingthe catalytic divalent cations (Xu, R. X. et al. Science 288:1822-1825(2000)).

For an allosterically regulated enzyme, such as glycogen phosphorylaseB, the site of interest includes all residues in the substrate bindingregion, residues in contact with the natural allosteric inhibitorglucose-6-phosphate, and residues in novel allosteric sites such asthose identified in binding other inhibitors such as CP320626(Oikonomakos N G, et al. Structure Fold Des 8:575-584 (2000)).

The TBM's either contain, or are modified to contain, a reactive residueat or near a site of interest. Preferably, the TBM's contain or aremodified to contain a thiol-containing amino acid residue at or near asite of interest. In this case, after a TBM is selected, the site ofinterest is calculated. Once the site of interest is known, a process ofdetermining which amino acid residue within, or near, the site ofinterest to modify is undertaken. For example, one preferredmodification results in substituting a cysteine residue for anotheramino acid residue located near the site of interest.

The choice of which residue within, or near, the site of interest tomodify is determined based on the following selection criteria. First, athree dimensional description of the TBM is obtained from one of severalwell-known sources. For example, the tertiary structure of many TBMs hasbeen determined through x-ray crystallography experiments. These x-raystructures are available from a wide variety of sources, such as theProtein Databank (PDB) which can be found on the Internet athttp://www.rcsb.org. Tertiary structures can also be found in theProtein Structure Database (PSdb) which is located at the PittsburgSupercomputer Center at http://www.psc.com.

In addition, the tertiary structure of many proteins, and proteincomplexes, has been determined through computer-based modelingapproaches. Thus, models of protein three-dimensional conformations arenow widely available.

Once the three dimensional structure of the TBM is known, a measurementis made based on a structural model of the wild type, or a variant form,of the target biological molecule from any atom of an amino acid withinthe site of interest across the surface of the protein for a distance ofapproximately 10 angstroms. Variant, which have been modified to containthe desired reactive groups (e.g. thiol groups, or thiol-containingresidues) are based on the identification of one or more wild-type aminoacid(s) on the surface of the target biological molecule that fallwithin that approximate 10-angstrom radius from the site of interest.For the purposes of this measurement, any amino acid having at least oneatom falling within the about 10 angstrom radius from any atom of anamino acid within the site of interest is a potential residue to bemodified to a thiol containing residue.

Preferred residues for modification are those that aresolvent-accessible. Solvent accessibility may be calculated fromstructural models using standard numeric (Lee, B. & Richards, F. M. J.Mol. Biol 55:379-400 (1971); Shrake, A. & Rupley, J. A. J. Mol. Biol.79:351-371 (1973)) or analytical (Connolly, M. L. Science 221:709-713(1983); Richmond, T. J. J. Mol. Biol. 178:63-89 (1984)) methods. Forexample, a potential cysteine variant is considered solvent-accessibleif the combined surface area of the carbon-beta (CB), or sulfur-gamma(SG) is greater than 21 Å² when calculated by the method of Lee andRichards (Lee, B. & Richards, F. M. J. Mol. Biol 55:379-400 (1971)).This value represents approximately 33% of the theoretical surface areaaccessible to a cysteine side-chain as described by Creamer et al.(Creamer, T. P. et al. Biochemistry 34:16245-16250 (1995)).

It is also preferred that the residue to be mutated to cysteine, oranother thiol-containing amino acid residue, not participate inhydrogen-bonding with backbone atoms or, that at most, it interacts withthe backbone through only one hydrogen bond. Wild-type residues wherethe side-chain participates in multiple (>1) hydrogen bonds with otherside-chains are also less preferred. Variants for which all standardrotamers (chi1 angle of −60°, 60°, or 180°) can introduce unfavorablesteric contacts with the N, CA, C, O, or CB atoms of any other residueare also less preferred. Unfavorable contacts are defined as interatomicdistances that are less than 80% of the sum of the van der Waals radiiof the participating atoms.

Wild-type residues that fall within highly flexible regions of theprotein are less preferred. Within structures derived from x-ray data,highly flexible regions can be defined as segments where the backboneatoms possess weak electron density or high temperature factors (>4standard deviations above the mean temperature factor for thestructure). Within structures derived from NMR data, highly flexibleregions can be defined as segments possessing <5 experimental restraints(derived from distance, dihedral coupling, and H-bonding data) perresidue, or regions displaying a high variability (>2.0 A² RMSdeviation) among the models in the ensemble. Additionally, residuesfound on convex “ridge” regions adjacent to concave surfaces are morepreferred while those within concave regions are less preferred cysteineresidues to be modified. Convexity and concavity can be calculated basedon surface vectors (Duncan, B. S. & Olson, A. J. Biopolymers 33:219-229(1993)) or by determining the accessibility of water probes placed alongthe molecular surface (Nicholls, A. et al. Proteins 11:281-296 (1991);Brady, G. P., Jr. & Stouten, P. F. J. Comput. Aided Mol. Des. 14:383-401(2000)). Residues possessing a backbone conformation that is nominallyforbidden for L-amino acids (Ramachandran, G. N. et al. J. Mol. Biol.7:95-99 (1963); Ramachandran, G. N. & Sasisekharahn, V. Adv. Prot. Chem.23:283-437 (1968)) are less preferred targets for modification to acysteine. Forbidden conformations commonly feature a positive value ofthe phi angle.

Other preferred variants are those which, when mutated to cysteine andlinked via a disulfide bond to an alkyl tether, would possess aconformation that directs the atoms of that tether towards the site ofinterest. Two general procedures can be used to identify these preferredvariants. In the first procedure, a search is made of unique structures(Hobohm, U. et al. Protein Science 1:409-417 (1992)) in the ProteinDatabank (Berman, H. M. et al. Nucleic Acids Research 28:235-242 (2000))to identify structural fragments containing a disulfide-bonded cysteineat position j in which the backbone atoms of residues j−1, j, and j+1 ofthe fragment can be superimposed on the backbone atoms of residues i−1,i, and i+1 of the target molecule with an RMSD of less than 0.75 A². Iffragments are identified that place the CB atom of the residuedisulfide-bonded to the cysteine at position j closer to any atom of thesite of interest than the CB atom of residue i (when mutated tocysteine), position i is considered preferred. In an alternativeprocedure, the residue at position i is computationally “mutated” to acysteine and capped with an S-Methyl group via a disulfide bond.

Further details of identifying site(s) of interest on the targets of theinvention are provided in co-pending application Ser. No. 60/310,725,filed on Aug. 7, 2001, the entire disclosure of which is herebyexpressly incorporated by reference.

4. Small Molecule Extender (SME)

(A) Static SME

In one embodiment of the invention the SME forms a “static” orirreversible covalent bond through the nucleophile or electrophile,preferably nucleophile, on the TBM, thereby forming an irreversibleTBM-SME complex. This method is illustrated in FIG. 2. Optionally theSME also forms a non-covalent bond with a first site of interest on theTBM. Additionally the SME contains a second functional group capable offorming a reversible bond with a library member of a library of smallorganic molecules, each molecule having a functional group capable offorming a reversible bond with the second functional group of the SME.The TBM-SME complex and library are subjected to conditions wherein thelibrary member having affinity, preferably the highest affinity, for thesecond site of interest on the TBM forms a reversible bond with theTBM-SME complex.

Preferred TBM's are proteins and the preferred nucleophiles on the TBM'ssuitable for forming an irreversible TBM-SME complex include —SH, —OH,—NH₂ and —COOH usually arising from side chains of cys, ser or thr, lysand asp or glu respectively. TBM's may be modified (e.g. mutants orderivatives) to contain these nucleophiles or may contain themnaturally. For example, cysteine proteases (e.g. Caspases, especially 1,3, 8 and 9; Cathesepins, especially S and K etc.) and phosphotases (e.g.PTPα, PTP1B, LAR, SHP1,2, PTPβ and CD45) are examples of suitableproteins containing naturally occurring cystiene thiol nucleophiles.Derivatizing such TBM's with a SME to produce a static TBM-SME complexand its reaction with a library member is illustrated below.

Here, the nucleophile on the TBM is the sulfur of a thiol, usually acysteine, which is reacted with 2, a SME containing a substituent Gcapable of forming an irreversible (under conditions that do notdenature the target) covalent bond and a free thiol, protected thiol orderivatized thiol SR′. Preferably G is a group capable of undergoingSN2-like attack by the thiol or forming a Michael-type adduct with thethiol to produce the irreversible reaction product 3 of that attackhaving a new covalent linkage —SG′-. The following are representativeexamples of G groups capable of undergoing SN2-like or Michael-typeaddition.

1) α-halo acids: F, Cl and Br substituted α to a COOH, PO₃H₂ or P(OR)O₂Hacid that is part of the SME can form a thioether with the thiol of theTBM. Simple examples of such a G-SME-SR′ are;

where X is the halogen and R′ is H, SCH₃, S(CH₂)_(n)A, where A is OH,COOH, SO₃H, CONH₂ or NH₂ and n is 2 or 3.

2) Fluorophos(phon)ates: These can be Sarin-like compounds which reactreadily with both SH and OH nucleophiles. For example, cys 215 of PTP1Bcan be reacted with a simple G-SME-SR′ represented by the following:

Here the phenyl ring represents a simplified SME, R is a substituted orunsubstituted loweralkyl and R′ is as defined above. These compoundsform thiophos(phon)ate SME's with the thiol nucleophile. These compoundsalso are capable of forming static TBM-SME's with naturally occurring—OH from serine or threonine phosphatases or β-lactamases.

3) Epoxides, aziridines and thiiranes: SME's containing these reactivefunctional groups are capable of undergoing SN2 ring opening reactionswith —SH, —OH and —COOH nucleophiles. Preferred examples of the latterare aspartyl proteases like β-secretase (BASE). Preferred genericexamples of epoxides, aziridines and thiiranes are shown below.

Here, R′ is as defined above, R is usually H or lower alkyl and R″ islower alkyl, lower alkoxy, OH, NH₂ or SR′. In the case of thiiranes thegroup SR′ is optionally present because upon nucleophilic attack andring opening a free thiol is produced which may be used in thesubsequent extended tethering reaction.

4) Halo-methyl ketones/amides: These compounds have the form—(C═O)—CH₂—X. Where X may be a large number of good leaving groups likehalogens, N₂, O—R (Where R may be substituted or unsubstitutedheteroaryl, Aryl, alkyl, —(P═O)Ar₂, —N—O—(C═O) aryl/alkyl, —(C═O)aryl/alkyl/alkylaryl and the like), S-Aryl, S-heteroaryl and vinylsulfones.

Fluromethylketones are simple examples of this class of activatedketones which result in the formation of a thioether when reacted with athiol containing protein. Other well known examples includeacyloxymethyl ketones like benzoyloxymethyl ketone, aminomethyl ketoneslike phenylmethylaminomethyl ketone and sulfonylaminomethyl ketones.These and other types of suitable compounds are reviewed in J. Med.Chem. 43(18) p 3351-71, Sep. 7, 2000.

5) Electrophilic aromatic systems: Examples of these include7-halo-2,1,3-benzoxadiazoles and ortho/para nitro substitutedhalobenzenes.

Compounds of this type form arylalkylthioethers with TBM's containing athiol.

6) Other suitable SN2 like reactions suitable for formation of staticcovalent bonds with TBM nucleophiles include formation of a Schiff basebetween an aldehyde and the amine group of lysine an enzymes like DNArepair proteins followed by reduction with for example NaCNBH₄.

7) Michael-type additions: Compounds of the form —RC═CR-Q, or —C≡C-Qwhere Q is C(═O)H, C(═O)R (including quinines), COOR, C(═O)NH₂,C(═O)NHR, CN, NO₂, SOR, SO₂R, where each R is independently substitutedor unsubstituted alkyl, aryl, hydrogen, halogen or another Q can formMichael adducts with SR (where R is H, glutathione or S-loweralkylsubstituted with NH₂ or OH), OH and NH₂ on the TBM.

8) Boronic acids: These compounds can be used to label ser or thrhydroxyls to form TBM-SME complexes of the form shown below:

where R′ is as defined above

In each of the foregoing cases a “static” or irreversible covalent bondis formed through the nucleophile on the TBM producing an irreversibleTBM-SME complex containing a thiol or protected thiol. These complexesare then exposed to a library of thiol or disulfide containing organiccompounds in the presence of a reducing agent (e.g. mercaptoethanol) forselection of a small molecule ligand capable of binding a second bindingsite on the TBM.

As noted above, in this static approach, the SME may, but does not haveto, include a portion that has binding affinity (i.e. is capable ofbonding to) a first site of interest on the TBM. Even if the SME doesnot include such portion, it must be of appropriate length andflexibility to ensure that the ligand candidates have free access to thesecond site of interest on the target.

(B) Dynamic SME

In another embodiment of the invention the SME is a double reversiblecovalent bond SME (“double disulfide” extender), that is, this SME isbifunctional and contains two functional groups (usually disulfide)capable of forming reversible covalent bonds. This SME forms a “dynamic”or first reversible covalent bond through a first functional group onthe SME with the nucleophile on the TBM, thereby forming a reversibleTBM-SME complex (7 below). Optionally the SME also forms a non-covalentbond with a first site of interest on the TBM (the portion of the SMEthat forms a non-covalent bond with the TBM is referred to herein asSME′). Additionally the SME contains or is modified to contain a secondfunctional group capable of forming a second reversible bond with alibrary member of a second library of small organic molecules, eachmolecule having a functional group capable of forming a reversible bondwith the first or second functional group of the SME. The TBM-SMEcomplex and the second library are subjected to conditions wherein thelibrary member having the highest affinity for a second site of intereston the TBM forms a reversible bond with the TBM-SME complex (8 below).Preferably the covalent bonds are disulfides, which may be reversible inthe presence of a reducing agent.

The dynamic extended tethering process is illustrated in FIG. 3 where aTMB containing or modified to contain a thiol or protected thiol isincubated with a first library of small organic molecules containing athiol or protected thiol (a disulfide-containing monophore) underconditions, such as with a reducing agent, wherein at least one memberof the library forms a disulfide bond linking the selected librarymember with the TBM. Optionally this process is repeated with a libraryof TBM's differing from one another by the location of the thiol orprotected thiol, i.e. different cysteine mutants of the same protein.Preferably each member of the small molecule library differs inmolecular weight from each of the other library members. Preferably thesmall molecule library contains from 1-100 members, more preferable from5-15 and most preferably about 10 members. Optionally the selected smallmolecule library member (selected monophore) also forms a noncovalentbond with a first site of interest on the TBM. The selected monophore,or a derivative thereof, is then modified to contain a second thiol orprotected thiol thereby forming a “double disulfide” extender. Thissynthetic double disulfide extender is then incubated with the TBM inthe presence of a second library of small organic molecules containing athiol or protected thiol (the library may be the same or different fromthe first library) under conditions, such as with a reducing agent likemercaptoethanol, wherein at least one member of the second library formsa disulfide bond linking the selected library member with the TBMthrough the double disulfide extender as shown in 8 above. Optionallythereafter a diaphore is synthesized based on the two selected librarymembers (monophores).

Two basic strategies exist for synthesizing a “double disulfide”extender. In the first, synthesis of the dynamic extender proceedsgenerically, that is by modification of the monophore linker without anymodification of the portion of the monophore that forms a non-covalentbond with the TBM. By way of illustration, the extender usually arisesfrom the screening of a disulfide monophore library as shown in FIG. 3.A typical monophore selected from the library or pool will contain alinker of 2 or 3 methylene units between the disulfide that links themonophore to the TBM cysteine and the portion of the monophore thatbinds non-covalently to the first site of interest on the TBM. Thismonophore linker can be derivatized as shown below to produce a doubledisulfide extender in which the “R” or variable group of the monophoreremains invariant and becomes the portion of the extender (SME′) thatbinds non-covalently with the first site of interest on the TBM.

Here the monophore is derivatized either at the methylene nearest thecysteamine nitrogen to produce dynamic double disulfide extender 1 or atthe cysteamine nitrogen itself to produce the symmetrical dynamic doubledisulfide extender 2.

Alternatively, when the monophore is a 3-mercaptopropionic acidderivative the alpha carbon can be derivatized to produce a genericdynamic double disulfide extender of the form shown in 3 below.

Optionally the amide nitrogen may be derivatized with an acyl orsulfonyl to produce an extender of the form shown in 4 above.

A second strategy involves derivatizing the portion of the monophorethat binds non-covalently to the first site of interest on the TBM. Thederivatization is preferably carried out at a site that minimally altersthe binding of the monophore to the first site of interest asillustrated below.

Here the dynamic tether is shown bound to the TBM thiol forming theTBM-SME complex, where R′ is the cysteamine radical. This complex canthen be contacted with a disulfide monophore or library of disulfidemonophores to obtain a linked compound having a higher affinity for theTBM than either the SME or selected monophore alone.

A second example of a SME designed form a disulfide monophore that bindsto the TBM is shown below. This dynamic SME can be contacted with theTBM in the presence of one or more disulfide monophores to form acovalent TBM-SME-monophore complex where the SME has an affinity for thefirst site of interest and the monophore has an affinity for the secondsite of interest on the TBM.

Detection and identification of the structure of the TBM-SME-monophorecomplex can be carried out by mass spectrometry or inhibition in afunctional assay (e.g. ELISA, enzyme assay etc.).

SME's are often customized for a particular TBM or family of TBM's. Forexample quinazoline derivatives are capable of forming static or dynamicextenders with the EGF receptor or an “RD” kinase. In the case of theEGF receptor, cys 773 is a suitable nucleophile for either a static ordynamic quinazoline extender as shown below;

where R¹ is linked to cys 773 through a Michael acceptor or disulfide,

-   -   R′0 is selected from    -   R² is —(CH₂)_(n)—SR′ and ═C(═O)—(CH₂)_(n)—SR′;    -   R³, R⁴ and R⁵ are —O—(CH₂)_(n)—SR′ and —(CH₂)_(n)—SR′;    -   R⁶ are; —(CH₂)_(n)—SR′; where n is 1, 2, or 3 and    -   R′ is H, a disulfide or a thiol protecting group.

Phosphotyrosine (P-tyr), phosphoserine (P-ser) and phoshpothreonine(P-thr) mimetics or surrogates may be used as extenders in the presentinvention to identivy fragments that interact with subsites nearby toimprove specificity or affinity for a target phosphotase. Thus extendedtethering using known substrates or inhibitors as “anchors” to findnearby fragments by standard covalent tethering with the extender is onepreferred embodiment of the instant invention.

Phosphotyrosine (P-tyr) mimetics are examples of SME's that may becustomized for phosphotases like PTP-1B, LAR etc. Known PTP-1B P-tyrmimetics derivitized with mercapto-propanoic acid and/or cystaeamine orthe protected forms thereof, shown below, bind to the active site of aPTP-1B cys mutant.

Such a compound may be used as a dynamic extender to select a secondfragment by covalent tethering as described above. The compound shownabove when bound to the target and titrated against β-mercaptoethanol(BME) displays a BME₅₀ (the concentration of β-mercaptoethanol that, atequalibrium, is capable of displacing 50% of the bound compound from thetarget) of about 2.5 mM. When using a dynamic extender it is preferredto measure the BME₅₀ for the dynamic extender and to screen for a secondfragment by covalent tethering at a total thiol concentration(BME+library thiols) at or below the BME₅₀ of the dynamic extender. Forexample, with the dynamic extender shown above having a BME₅₀ of 2.5 mM,the total thiol concentration in the second fragment screening stepshould be 2.5 mM or less and more preferrabley about 2 fold less, e.g.about 1 mM or less. Alternatively the dynamic extenter may be convertedto a static extender removing the second fragment screening total thiolconcentration issue. When converting a dynamic extender to a staticextender it is important to maintain the same atom count so thatnon-covalent binding of the static extender to the target will not bedistorted. For similar reasons it is important to minimize introductionof other other bulky atoms or groups. With these factors in mind, theabove dynamic extender may be converted into the static extendersdefined below.

where R¹ is selected from

and where R² is selected from

In still another embodiment of the invention, an extender may be apeptide either reversibly or irreversibly bound to the TBM. In thisembodiment the peptide is from about 2-15 residues long, preferable from5 to 10 residues, and may be composed of natural and/or artificial alphaamino acids. An example of such a peptide extender is an alpha helicalp53 fragment peptide (or smaller known non-natural peptides) that arecapable of binding to the N-terminal domain of MDM2 in a deephydrophobic cleft with nM affinities. BCL-2 and BCL-xL are also known tocontain deep peptide-binding grooves analogous the MDM2. Peptides thatbind to these targets may also be useful peptide extenders according tothe present invention. For example, a fragment peptide of p53 may form areversible (e.g. disulfide) bond through an existing (e.g. cys) thiol oran introduced thiol (introduced cys, cysteamine derivatized with thecarboxyl terminus or mercapto-propanoic acid through the amino terminus)on the peptide with an existing or introduced thiol on the TBM. In thiscase a TBM-peptide extender complex will be formed which is capable ofbeing used to select a thiol or disulfide fragment from a subsequentcovalent tether screen. This dynamic peptide extender will have oneother free or protected thiol (e.g. one of the above not used to formthe TBM-peptide extender complex), which is contacted with a library ofthiol or protected thiol fragments under conditions suitable for forminga covalent disulfide bond with a fragment having affinity for the TBM.Optionally the peptide extender may be a static one where anirreversible covalent bond is formed with a nucleophile or electrophileon the TBM as described above. Optionally in this embodiment, aphotoaffinity label may be used to attach the peptide extender to theTBM. As above, a free or protected thiol pre-existing or introduced isused to form a disulfide in a subsequent screen to find a small moleculefragment having affinity for the TBM.

Such a peptide extender may also be a synthetic peptide such as the“Z-WQPY” peptide where the TBM is the IL-1 receptor. Here, the peptideFEWTPGYWQPYALPL or fragments, mutants or analogues thereof can be usedas a static or dynamic extender as described above to discover fragmentsvia covalent tethering, where the disulfide tether is to or with theextender and the non-covalent bond is between the selected fragment andthe TBM.

Other chemistries available for forming a reversible or irreversiblecovalent bond between reactive groups on a SME and a target or ligand,respectively, or between two ligands, are well known in the art, and aredescribed in basic textbooks, such as, e.g. March, Advanced OrganicChemistry, John Wiley & Sons, New York, 4^(th) edition, 1992. Reductiveaminations between aldehydes and ketones and amines are described, forexample, in March et al., supra, at pp. 898-900; alternative methods forpreparing amines at page 1276; reactions between aldehydes and ketonesand hydrazide derivatives to give hydrazones and hydrazone derivativessuch as semicarbazones at pp. 904-906; amide bond formation at p. 1275;formation of ureas at p. 1299; formation of thiocarbamates at p. 892;formation of carbamates at p. 1280; formation of sulfonamides at p.1296; formation of thioethers at p. 1297; formation of disulfides at p.1284; formation of ethers at p. 1285; formation of esters at p. 1281;additions to epoxides at p. 368; additions to aziridines at p. 368;formation of acetals and ketals at p. 1269; formation of carbonates atp. 392; formation of denamines at p. 1264; metathesis of alkenes at pp.1146-1148 (see also Grubbs et al., Acc, Chem. Res. 28:446-453 [1995]);transition metal-catalyzed couplings of aryl halides and sulfonates withalkanes and acetylenes, e.g. Heck reactions, at p.p. 717-178; thereaction of aryl halides and sulfonates with organometallic reagents,such as organoboron, reagents, at p. 662 (see also Miyaura et al., Chem.Rev. 95:2457 [1995]); organotin, and organozinc reagents, formation ofoxazolidines (Ede et al., Tetrahedron Letts. 28:7119-7122 [1997]);formation of thiazolidines (Patek et al., Tetrahedron Letts.36:2227-2230 [1995[); amines linked through amidine groups by couplingamines through imidoesters (Davies et al., Canadian J.Biochem.c50:416-422 [1972]), and the like. In particular,disulfide-containing small molecule libraries may be made fromcommercially available carboxylic acids and protected cysteamine (e.g.mono-BOC-cysteamine) by adapting the method of Parlow et al., Mol.Diversity 1:266-269 (1995), and can be screened for binding topolypeptides that contain, or have been modified to contain, reactivecysteines. All of the references cited in this section are herebyexpressly incorporated by reference.

While it is usually preferred that the attachment of the SME does notdenature the target, the TBM-SME complex may also be formed underdenaturing conditions, followed by refolding the complex by methodsknown in the art. Moreover, the SME and the covalent bond should notsubstantially alter the three-dimensional structure of the target, sothat the ligands will recognize and bind to a site of interest on thetarget with useful site specificity. Finally, the SME should besubstantially unreactive with other sites on the target under thereaction and assay conditions.

5. Detection and Identification of Ligands Bound to a Target

The ligands bound to a target can be readily detected and identified bymass spectroscopy (MS). MS detects molecules based on mass-to-chargeratio (m/z) and thus can resolve molecules based on their sizes(reviewed in Yates, Trends Genet. 16: 5-8 [2000]). A mass spectrometerfirst converts molecules into gas-phase ions, then individual ions areseparated on the basis of m/z ratios and are finally detected. A massanalyzer, which is an integral part of a mass spectrometer, uses aphysical property (e.g. electric or magnetic fields, or time-of-flight[TOF]) to separate ions of a particular m/z value that then strikes theion detector. Mass spectrometers are capable of generating data quicklyand thus have a great potential for high-throughput analysis. MS offersa very versatile tool that can be used for drug discovery. Massspectroscopy may be employed either alone or in combination with othermeans for detection or identifying the organic compound ligand bound tothe target. Techniques employing mass spectroscopy are well known in theart and have been employed for a variety of applications (see, e.g.,Fitzgerald and Siuzdak, Chemistry & Biology 3: 707-715 [1996]; Chu etal., J. Am. Chem. Soc. 118: 7827-7835 [1996]; Siudzak, Proc. Natl. Acad.Sci. USA 91: 11290-11297 [1994]; Burlingame et al., Anal. Chem. 68:599R-651R [1996]; Wu et al., Chemistry & Biology 4: 653-657 [1997]; andLoo et al., Am. Reports Med. Chem. 31: 319-325 [1996]).

However, the scope of the instant invention is not limited to the use ofMS. In fact, any other suitable technique for the detection of theadduct formed between the biological target molecule and the librarymember can be used. For example, one may employ various chromatographictechniques such as liquid chromatography, thin layer chromatography andlikes for separation of the components of the reaction mixture so as toenhance the ability to identify the covalently bound organic molecule.Such chromatographic techniques may be employed in combination with massspectroscopy or separate from mass spectroscopy. One may optionallycouple a labeled probe (fluorescently, radioactively, or otherwise) tothe liberated organic compound so as to facilitate its identificationusing any of the above techniques. In yet another embodiment, theformation of the new bonds liberates a labeled probe, which can then bemonitored. A simple functional assay, such as an ELISA or enzymaticassay may also be used to detect binding when binding of the extender orsecond fragment to the target occurs in an area essential for what theassay measures (e.g. binding to a “Hot Spot” in a protein:protein ELISAor binding in the substrate binding pocket for an enzyme assay). Othertechniques that may find use for identifying the organic compound boundto the target molecule include, for example, nuclear magnetic resonance(NMR), capillary electrophoresis, X-ray crystallography, and the like,all of which will be well known to those skilled in the art.

6. Preparation of Conjugate Molecules (e.g. Diaphores)

Linker elements that find use for linking two or more organic moleculeligands to produce a conjugate molecule will be multifunctional,preferably bifunctional, cross-linking molecules that can function tocovalently bond at least two organic molecules together via reactivefunctionalities possessed by those molecules. Linker elements will haveat least two, and preferably only two, reactive functionalities that areavailable for bonding to at least two organic molecules, wherein thosefunctionalities may appear anywhere on the linker, preferably at eachend of the linker and wherein those functionalities may be the same ordifferent depending upon whether the organic molecules to be linked havethe same or different reactive functionalities. Linker elements thatfind use herein may be straight-chain, branched, aromatic, and the like,preferably straight chain, and will generally be at least about 2 atomsin length, more generally more than about 4 atoms in length, and oftenas many as about 12 or more atoms in length. Linker elements willgenerally comprise carbon atoms, either hydrogen saturated orunsaturated, and therefore, may comprise alkanes, alkenes or alkynes,and/or other heteroatoms including nitrogen, sulfur, oxygen, and thelike, which may be uhsubstituted or substituted, preferably with alkyl,alkoxyl, hydroxyalkyl or hydroxyalkyl groups. Linker elements that finduse will be a varying lengths, thereby providing a means for optimizingthe binding properties of a conjugate ligand compound preparedtherefrom. The first organic compound that covalently bound to thetarget biomolecule may itself possess a chemically reactive group thatprovides a site for bonding to a second organic compound. Alternatively,the first organic molecule may be modified (either chemically, bybinding a compound comprising a chemically reactive group thereto, orotherwise) prior to screening against a second library of organiccompounds.

7. Compounds of the Invention

The compounds of the present invention are characterized by encompassingat least one, preferably at least two, ligands at least one of which hasbeen identified by the extended tethering approach disclosed herein, andanalogs of such compounds. Accordingly, the compounds of the presentinvention encompass numerous chemical classes, including but not limitedto small organic molecules, peptides, (poly)nucleotides,(oligo)saccharides, etc. The ligands identified by the present methodstypically serve as lead compounds for the development of furthervariants and derivatives designed by following well known techniques. Inparticular, the ligands identified (including monophores, diaphores, andmore complex structures) are amenable to medicinal chemistry andaffinity maturation, and can be rapidly optimized using structure-aideddesign. The present extended tethering approach is superior over otherknown techniques, including combinatorial chemistry, in that it allowsfurther chemical modifications focused on ligands which have alreadybeen shown to to bind to different sites on a target, e.g. a TBM.

8. Uses of Compounds Identified

The method of the present invention is a powerful technique forgenerating drug leads, allows the identification of two or morefragments that bind weakly or with moderate binding affinity to a targetat sites near one another, and the synthesis of diaphores or largermolecules comprising the identified fragments (monophores) covalentlylinked to each other to produce higher affinity compounds. The diaphoresor similar multimeric, compounds including further ligand compounds, arevaluable tools in rational drug design, which can be further modifiedand optimized using medicinal chemistry approaches and structure-aideddesign.

The diaphores identified in accordance with the present invention andthe modified drug leads and drugs designed therefrom can be used, forexample, to regulate a variety of in vitro and in vivo biologicalprocesses which require or depend on the site-specific interaction oftwo molecules. Molecules which bind to a polynucleotide can be used, forexample, to inhibit or prevent gene activation by blocking the access ofa factor needed for activation to the target gene, or represstranscription by stabilizing duplex DNA or interfering with thetranscriptional machinery.

9. Pharmaceutical Compositions

The ligands identified in accordance with the present invention, andcompounds comprising such ligands, as well as analogues of suchcompounds, can be used in pharmaceutical compositions to prevent and/ortreat a targeted disease or condition. The target disease or conditiondepends on the biological/physiological function of the target, e.g. TBMto which the ligand or the compounds designed based on such ligand(s)binds. Examples of such diseases and conditions are listed in the tableof TBM's above.

Suitable forms of pharmaceutical compositions, in part, depend upon theuse or route of entry, for example oral, transdermal, inhalation, or byinjections. Such forms should allow the agent or composition to reach atarget cell whether the target cell is present in a multicellular hostor in culture. For example, pharmacological agents or compositionsinjected into the blood stream should be soluble. Other factors areknown in the art, and include considerations such as toxicity and formsthat prevent the agent or composition from exerting its effect.

The active ingredient, when appropriate, can also be formulated aspharmaceutically acceptable salts (e.g., acid addition salts) and/orcomplexes. Pharmaceutically acceptable salts are non-toxic at theconcentration at which they are administered. Pharmaceuticallyacceptable salts include acid addition salts such as those containingsulfate, hydrochloride, phosphate, sulfonate, sulfamate, sulfate,acetate, citrate, lactate, tartarate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate, cyclohexylsulfonate,cyclohexylsulfamate an quinate.

Pharmaceutically acceptable salts can be obtained from acids such ashydrochloric acid, sulfuric acid, phosphoric acid, sulfonic acid,sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid,malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonicacid, p-toluenesulfonic acid, cyclobexylsulfonic acid,cyclohexylsulfamic acid, and quinic acid. Such salts may be prepared by,for example, reacting the free acid or base forms of the product withone or more equivalents of the appropriate base or acid in a solvent ormedium in which the salt is insoluble, or in a solvent such as water,which is then removed in vacuo or by freeze-drying or by exchanging theions of an existing salt for another ion on a suitable ion exchangeresin.

Carriers or excipients can also be used to facilitate administration ofthe compound. Examples of carriers and excipients include calciumcarbonate, calcium phosphate, various sugars such as lactose, glucose,or sucrose, or types of starch, cellulose derivatives, gelatin,vegetable oils, polyethylene glycols and physiologically compatiblesolvents. The compositions or pharmaceutical compositions can beadministered by different routes including, but not limited to,intravenous, intra-arterial, intraperitoneal, intrapericardial,intracoronary, subcutaneous, intramuscular, oral topical, ortransmucosal.

The desired isotonicity of the compositions can be accomplished usingsodium chloride or other pharmaceutically acceptable agents such asdextrose, boric acid, sodium tartarate, propylene glycol, polyols (suchas mannitol and sorbitol), or other inorganic or organic solutes.

Techniques and ingredients for making, pharmaceutical formulationsgenerally may be found, for example, in Remington's PharmaceuticalSciences, 18^(th) Edition, Mack Publishing Co., Easton, Pa. 1990. Seealso, Wang and Hanson “Parental Formulations of Proteins and Peptides:Stability and Stabilizers,” Journal of Parental Science and technology,Technical Report No. 10, Supp. 42-2S (1988). A suitable administrationformat can be best determined by a medical practitioner for each diseaseor condition individually, and also in view of the patient's condition.

Pharmaceutical compositions are prepared by mixing the ingredientsfollowing generally accepted procedures. For example, the selectedcomponents can be mixed simply in a blender or other standard device toproduce a concentrated mixture which can then be adjusted to the finalconcentration and viscosity by the addition of water or thickening agentand possibly a buffer to control pH or an additional solute to controltonicity.

The amounts of various compounds for use in the compositions of theinvention to be administered can be determined by standard procedures.Generally, a therapeutically effective amount is between about 100 mg/kgand 10⁻¹² mg/kg depending on the age and size of the patient, and thedisease or disorder associated with the patient. Generally, it is anamount between about 0.05 and 50 mg/kg of the individual to be treated.The determination of the actual dose is well within the skill of anordinary physician.

10. Description of Preferred Embodiments

In a preferred embodiment, the methods of the present invention are usedto identify low molecular weight ligands that bind to at least twodifferent sites of interest on target proteins through intermediarydisulfide tethers formed between a first ligand and the protein, and areactive group on the first ligand and a second ligand, respectively.

The low molecular weight ligands screened in preferred embodiments ofthe invention will be, for the most part, small chemical molecules thatwill be less than about 2000 daltons in size, usually less than about1500 daltons in size, more usually less than about 750 daltons in size,preferably less than about 500 daltons in size, often less than about250 daltons in size, and more often less than about 200 daltons in size,although organic molecules larger than 2000 daltons in size will alsofind use herein. In one preferred embodiment, such small chemicalmolecules are small organic molecules, other than polypeptides orpolynucleotides. In another preferred embodiment, the small organicmolecules are non-polymeric, i.e. are not peptide, polypeptides,polynucleotides, etc.

Organic molecules may be obtained from a commercial or non-commercialsource. For example, a large number of small organic chemical compoundsare readily obtainable from commercial suppliers, such as AldrichChemical Co., Milwaukee, Wis., and Sigma Chemical Co., Sr. Louis, Mo.,or may be obtained by chemical synthesis. The methods of the presentinvention are preferably used to screen libraries of small organiccompounds carrying appropriate reactive group, preferably thiol orprotected thiol groups.

In recent years, combinatorial libraries, typically having from dozensto hundreds of thousands of members, have become a major tool for liganddiscovery and drug development. In general, libraries of organiccompounds which find use herein will comprise at least 2 organiccompounds, often at least about 25 different organic compounds, moreoften at least about 100 different organic compounds, usually at leastabout 300 different organic compounds, preferably at least about 2500different organic compounds, and most preferably at least about 5000 ormore different organic compounds. Populations may be selected orconstructed such that each individual molecule of the population may bespatially separated from the other molecules of the population (e.g. inseparate microtiter well) or two or more members of the population maybe combined if methods for deconvolution are readily available. Usually,each member of the organic molecule library will be of the same chemicalclass (i.e. all library members are aldehydes, all library members areprimary amines, etc.), however, libraries of organic compounds may alsocontain molecules from two or more different chemical classes.

In a preferred embodiment, the target biological molecule (TBM) is apolypeptide that contains or has been modified to contain a thiol group,protected thiol group or reversible disulfide bond. The TBM is thenreacted with a Small Molecule extender (SME), which includes a portionhaving affinity for a first site of interest on the TBM and a groupreactive with the thiol, protected thiol or reversible disulfide bond onthe TBM. As discussed above, the linkage between the TBM and the SME maybe either an irreversible covalent bond (“static” extended tethering),or a reversible covalent bond (“dynamic” extended tethering) to form aTBM-SME complex. Whether the static or dynamic approach is used, theTBM-SME complex is then used to screen a library of disulfide-containingmonophores to identify a library member that has intrinsic affinity,most preferably the highest intrinsic affinity, for a second bindingsite (site of interest) on the target molecule. In a preferredembodiment, the reactive group on the modified TBM is a free-thiol groupcontributed by the extender, and the library is made up of smallmolecular weight compounds containing reactive thiol group. Fordisulfide tethering to capture the most stable ligand, the reaction mustbe under rapid exchange to allow for equilibration. In a preferredembodiment, the reaction is carried out in the presence of catalyticamount of a reducing agent such as 2-mercaptoethanol. Thermodynamicequilibrium reached in the presence of a reducing agent will favor theformation of disulfide bond between thiol group of the extender on themodified TBM and thiol group of a member of the library having intrinsicaffinity for the TBM. Thus, two different ligands with intrinsicaffinity for two different sites on the same TBM will be covalentlylinked to form a diaphore. The diaphore will bind to the TBM with ahigher affinity than any of the constituent monophore units. Themonophore units in a diaphore may be from the same or different chemicalclasses. By “same chemical class” is meant that each monophore componentis of the same chemical type, i.e., both are aldehyde or amines etc.

In a particular embodiment, the target can be present on a chipcontacted with the ligand candidates. In this case, the covalent bondlinking the first ligand to the target may be formed with the chip, inwhich case, the chip will become part of the covalent bond, representinga special class of “Small Molecule Extenders.”

The library of the ligand candidates, e.g. small organic moleculeligands, can be attached to a solid surface, e.g. displayed on beads,for example as described in PCT publication WO 98/11436 published onMar. 19, 1998. In a particular embodiment, beads are modified tointroduce reactive groups, e.g. a low level of sulfhyrdyl groups. Alibrary of ligand candidates is then synthesized on the modified beads.Subsequently, the library is incubated, under oxidizing conditions, withthe target containing or modified to contain a reactive group, e.g. asulfhydryl group such that s disulfide bond can be formed between thetarget and the sulfhydryl on the bead. The beads are then washed in thepresence of a reducing agent, followed by incubation in the presence ofa sulfhydryl quenching agent, such as iodoacetate. The beads may then bewashed under denaturing conditions to remove any non-covalently boundtarget.

EXAMPLES

The invention is further illustrated by the following, non-limitingexamples. Unless otherwise noted, all the standard molecular biologyprocedures are performed according to protocols described in (MolecularCloning: A Laboratory Manual, vols. 1-3, edited by Sambrook, J.,Fritsch, E. F., and Maniatis, T., Cold Spring Harbor Laboratory Press,1989; Current Protocols in Molecular Biology, vols. 1-2, edited byAusbubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J. G., Smith,J., and Struhl, K., Wiley Interscience, 1987).

The concept of basic tethering approach has been described by Erlansonet al., supra, and in PCT Publication No. WO 00/00823. The “extendedtethering” approach is illustrated in this application using caspase-3as a target biological molecule (TBM). Caspases are a family of cysteineproteases, that are known to participate in the initiation and executionof programmed cell death (apoptosis). The first caspase (now referred toas Caspase-1) was originally designated as interleukin-1β-convertingenzyme (ICE) (Thomburry et al., Nature 356:768-774 [1992]; Cerretti etal., Science 356:97-100 [1992]). Subsequently a large number of caspaseshave been identified and characterized forming a caspase family.Presently there are at least 10 members in the family (Caspase-1 toCaspase-10). Caspases are expressed in cells in an enzymaticallyinactive form and become activated by proteolytic cleavage in responseto an apoptotic stimulus. The inactive proenzyme form consists of alarge and a small domain (subunit), in addition to an inhibitoryN-terminal domain. Caspase activation involves the processing of theproenzyme into the large and small subunits, which occurs internallywithin the molecule. Caspases are activated either by self-aggregationand autoprocessing (as in the initiation of apoptosis), or via cleavageby an activated upstream caspase (as in the execution phase ofapoptosis). For review, see, for example, Cohen G. M. Biochem. J. 326:1-16 (1997).

Based on a known tetrapeptide inhibitor of caspase (Ator and Dolle,Current Pharmaceutical Design 1:191-210 (1995)), an extender wassynthesized: 2,6-Dichloro-benzoic acid3-(2-acetylsulfanyl-acetylamino)-4-carboxy-2-oxo-butyl ester (shown ascompound 5 in FIG. 4), the synthesis of which is described in Example 2below. A generic structure of extender is shown in FIG. 4. Caspase wasmodified by reacting with the extender (Example 3) and subsequently usedas a biological target molecule for screening of disulfide libraryprepared as described in Example 1, by using the extended tetheringapproach.

All commercially available materials were used as received. Allsynthesized compounds were characterized by ¹H NMR [Bruker (Billerica,Mass.) DMX400 MHz Spectrometer] and HPLC-MS (Hewlett-Packard Series 1100MSD).

Example 1 Disulfide Libraries

Disulfide libraries were synthesized using standard chemistry from thefollowing classes of compounds: aldehydes, ketones, carboxylic acids,amines, sulfonyl chlorides, isocyanates, and isothiocyanates. Forexample, the disulfide-containing library members were made fromcommercially available carboxylic acids andmono-N-(tert-butoxycarbonyl)-protected cystamine (mono-BOC-cystamine) byadapting the method of Parlow and coworkers (Parlow and Normansell, Mol.Diversity 1: 266-269 [1995]). Briefly, 260 μmol of each carboxylic acidwas immobilized onto 130 μmol equivalents of4-hydroxy-3-nitrobenzophenone on polystyrene resin using1,3-diisopropylcarbodiimide (DIC) in N,N-dimethylformamide (DMF). After4 h at room temperature, the resin was rinsed with DMF (×2),dichloromethane (DCM, ×3), and tetrahydrofuran (THF, ×1) to removeuncoupled acid and DIC. The acids were cleaved from the resin via amideformation with 66 μmol of mono-BOC protected cystamine in THF. Afterreaction for 12 h at ambient temperature, the solvent was evaporated,and the BOC group was removed from the uncoupled half of each disulfideby using 80% trifluoroacetic acid (TFA) in DCM. The products werecharacterized by HPLC-MS, and those products that were substantiallypure were used without further purification. A total of 530 compoundswere made by using this methodology.

Libraries were also constructed from mono-BOC-protected cystamine and avariety of sulfonyl chlorides, isocyanates, and isothiocyanates. In thecase of the sulfonyl chlorides, 10 μmol of each sulfonyl chloride wascoupled with 10.5 μmol of mono-BOC-protected cystamine in THF (with 2%diisopropyl ethyl amine) in the presence of 15 mg of poly(4-vinylpyridine). After 48 h, the poly(4-vinylpyridine) was removed viafiltration, and the solvent was evaporated. The BOC group was removed byusing 50% TFA in DCM. In the case of the isothiocyanates, 10 μmol ofeach isocyanate or isothiocyanate was coupled with 10.5 μmol ofmono-BOC-protected cystamine in THF. After reaction for 12 h at ambienttemperature, the solvent was evaporated, and the BOC group was removedby using 50% TFA in DCM. A total of 212 compounds were made by usingthis methodology.

Finally, oxime-based libraries were constructed by reacting 10 μmol ofspecific aldehydes or ketones with 10.5 μmol of HO(CH₂)₂S—S(CH₂)₂ONH₂ in1:1 methanol/chloroform (with 2% acetic acid added) for 12 h at ambienttemperature to yield the oxime product. A total of 448 compounds weremade by using this methodology.

Individual library members were redissolved in either acetonitrile ordimethyl sulfoxide to a final concentration of 50 or 100 mM. Aliquots ofeach of these were then pooled into groups of 8-15 discrete compounds,with each member of the pool having a unique molecular weight.

Example 2 Extender (SME) Synthesis

For extended tethering approach, extender (2,6-Dichloro-benzoic acid3-(2-acetylsulfanyl-acetylamino)-4-carboxy-2-oxo-butyl ester, shown ascompound 5 in FIG. 4) was synthesized using a series of chemicalreactions as shown in FIG. 4, and described below.

Synthesis of 2-(2-Acetylsulfanyl-acetylamino)-succinic acid 4-tert-butylester (compound 2, FIG. 4)

Acetylsulfanyl-acetic acid pentafluorophenyl ester (1.6 g, 5.3 mmol) andH-Asp(OtBu)-OH (1 g, 5.3 mmol) were mixed in 20 ml of drydichloromethane (DCM). Then 1.6 ml of triethylamine (11.5 mmol) wasadded, and the reaction was allowed to proceed at ambient temperaturefor 3.5 hours. The organic layer was then extracted with 3×15 ml of 1 Msodium carbonate, the combined aqueous fractions were acidified with 100ml of 1 M sodium hydrogensulfate and extracted with 3×30 ml ethylacetate. The combined organic fractions were then rinsed with 30 ml of 1M sodium hydrogensulfate, 30 ml of 5 M NaCl, dried over sodium sulfate,filtered, and evaporated under reduced pressure to yield 1.97 g of anearly colorless syrup which was used without further purification.MW=305 (found 306, M+1).

Synthesis of 3-(2-Acetylsulfanyl-acetylamino)-5-chloro-4-oxo-pentanoicacid tert-butyl ester (compound 3, FIG. 4)

The free acid (compound 2) was dissolved in 10 ml of dry tetrahydrofuran(THF), cooled to 0° C., and treated with 0.58 ml N-methyl-morpholine(5.3 mmol) and 0.69 isobutylchloroformate. Dense white precipitateimmediately formed, and after 30 minutes the reaction was filteredthrough a glass frit and transferred to a new flask with an additional10 ml of THF. Meanwhile, diazomethane was prepared by reacting1-methyl-3-nitro-1-nitrosoguanidine (2.3 g, 15.6 mmol) with 7.4 ml of40% aqueous KOH and 25 ml diethyl ether for 45 minutes at 0° C. Theyellow ether layer was then decanted into the reaction containing themixed anhydride, and the reaction allowed to proceed while slowlywarming to ambient temperature over a period of 165 minutes. Thereaction was cooled to 8° C., and 1.5 ml of 4 N HCl in dioxane (6 mmoltotal) was added dropwise. This resulted in much bubbling, and theyellow solution became colorless. The reaction was allowed to proceedfor two hours while gradually warming to ambient temperature and thenquenched with 1 ml of glacial acetic acid. The solvent was removed underreduced pressure and the residue redissolved in 75 ml ethyl acetate,rinsed with 2×50 ml saturated sodium bicarbonate, 50 ml 5 M NaCl, driedover sodium sulfate, filtered, and evaporated to dryness beforepurification by flash chromatography using 90:10 chloroform : ethylacetate to yield 0.747 g of light yellow oil (2.2 mmol, 42% from (1)).Expected MW=337.7, found 338 (M+1).

Synthesis of 2,6-Dichloro-benzoic acid3-(2-acetylsulfanyl-acetylamino)-4-tert-butoxycarbonyl-2-oxo-butyl ester(compound 4, FIG. 4)

The chloromethylketone (compound 3) (0.25 g, 0.74 mmol) was dissolved in5 ml of dry N,N-dimethylformamide (DMF), to which was added 0.17 g2,6-dichlorobenzoic acid (0.89 mmol) and 0.107 g KF (1.84 mmol). Thereaction was allowed to proceed at ambient temperature for 19 hours, atwhich point it was diluted with 75 ml ethyl acetate, rinsed with 2×50 mlsaturated sodium bicarbonated, 50 ml 1 M sodium hydrogen sulfate, 50 ml5 M NaCl, dried over sodium sulfate, filtered, and dried under reducedpressure to yield a yellow syrup which HPLC-MS revealed to be about 75%product and 25% unreacted (3). This was used without furtherpurification. Expected MW=492.37, found 493 (M+1).

Synthesis of 2,6-Dichloro-benzoic acid3-(2-acetylsulfanyl-acetylamino)-4-carboxy-2-oxo-butyl ester (compound5, FIG. 4)

The product of the previous step (compound 4) was dissolved in 10 ml ofdry DCM, cooled to 0° C., and treated with 9 ml trifluoroacetic acid(TFA). The reaction was then removed from the ice bath and allowed towarm to ambient temperature over a period of one hour. Solvent wasremoved under reduced pressure, and the residue redissoved twice in DCMand evaporated to remove residual TFA. The crude product was purified byreverse-phase high-pressure liquid chromatography to yield 101.9 mg(0.234 mmol, 32% from (3)) of white hygroscopic powder. ExpectedMW=436.37, found 437 (M+1). This was dissolved in dimethylsulfoxide(DMSO) to yield a 50 mM stock solution.

Example 3 Modification of Caspase 3 with Extender

Caspase 3 was cloned, overexpressed, and purified using standardtechniques (Rotonda et al., Nature Structural Biology 3(7):619-625(1996)). To 2 ml of a 0.2 mg/ml Caspase 3 solution was added 10 ml of 50mM 2,6-Dichloro-benzoic acid3-(2-acetylsulfanyl-acetylamino)-4-carboxy-2-oxo-butyl ester (compound5, FIG. 3) synthesized as described in Example 2, and the reaction wasallowed to proceed at ambient temperature for 3.5 hours, at which pointmass spectroscopy revealed complete modification of the caspase 3 largesubunit (MW 16861Da, calculated MW 16860Da). The thioester wasdeprotected by adding 0.2 ml of 0.5 M hydroxylamine buffered in PBSbuffer, and allowing the reaction to proceed for 18 hours, at whichpoint the large subunit had a mass of 16819Da (calculated 16818Da). Theprotein was concentrated in a Ultrafree 5 MWCO unit (Millipore) and thebuffer exchanged to 0.1 M TES pH 7.5 using a Nap-5 column (AmershamPharmacia Biotech). The structure of the resulting “extended” caspase-3is shown in FIG. 6.

The protein was then screened against a disulfide library prepared asdescribed above, in Example 1, and using the methodology described inExample 4 below.

Example 4 Screening of Disulfide Library

In a typical experiment, 1 μl of a DMSO solution containing a library of8-15 disulfide-containing compounds was added to 49 μl of buffercontaining extender-modified protein. When mass spectroscopy was usedfor the identification of the bound ligand, the compounds were chosen sothat each has a unique molecular weight. For example, these molecularweights differ by at least 10 atomic mass units so that deconvolution isunambiguous. Although pools of 8-15 disulfide-containing compounds weretypically chosen for screening because of the ease of deconvolution,larger pools can also be used. The protein was present at aconcentration of ˜15 μM, each of the disulfide library members waspresent at ˜0.2 mM, and thus the total concentration of all disulfidelibrary members was ˜2 mM. The reaction was done in a buffer containing25 mM potassium phosphate (pH 7.5) and 1 mM 2-mercaptoethanol, althoughother buffers and reducing agents can be used. The reactions wereallowed to equilibrate at ambient temperature for at least 30 min. Theseconditions can be varied considerably depending on the ease with whichthe protein ionizes in the mass spectrometer, the reactivity of thespecific cysteine(s), etc.

After equilibration of aspartyl-conjugated caspase-3 (Example 3) andlibrary (Example 1), the reaction was injected onto an HP1100 HPLC andchromatographed on a C₁₈ column attached to a mass spectrometer(Finnigan-MAT LCQ, San Jose, Calif.). The multiply charged ions arisingfrom the protein were deconvoluted with available software (XCALIBUR) toarrive at the mass of the protein. The identity of any library memberbonded through a disulfide bond to the protein was then easilydetermined by subtracting the known mass of the unmodified protein fromthe observed mass. This process assumes that the attachment of a librarymember does not dramatically change the ionization characteristics ofthe protein itself, a conservative assumption because in most cases theprotein will be at least 20-fold larger than any given library member.This assumption was confirmed by demonstrating that small moleculesselected by one protein are not selected by other proteins.

The results of a representative experiment are shown in FIG. 6. Thespectrum on the right side of FIG. 6 shows the result of reacting“extended” Casase-3 (synthesized as described in Example 3), with adisulfide-containing molecule identified from a pool as modifyingextended Caspase-3. The predominant peak obtained (mass of 17,094)corresponds to Caspase-3 covalently linked to the small molecule ligandwhich has an intrinsic affinity for a second site of interest onCaspase-3, resulting in the diaphore compound shown above the peak.

The mass spectrum shown on the left side is a deconvoluter mass spectrumof unmodified Caspase-3 (a cysteine-containing polypeptide target), andthe same disulfide-containing small molecule ligand used above. Thespectrum reveals a predominant peak corresponding to the mass ofunmodified Caspase-3 (16,614 DA). A significantly smaller peakrepresents Caspase-3 disulfide-bonded to 2-aminoethanethiol (combinedmass: 16,691 Da). Note that here the small molecule ligand is notselected because its binding site is too far from the reactive cysteineand no extender has introduced.

The initial lead compound, identified as describe above, was thenmodified in order to evaluate the relative importance of varioussubstituents in specific binding to Caspase-3.

All references cited throughout the specification are hereby expresslyincorporated by reference.

While the present invention has been described with reference to thespecific embodiment thereof, it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the object, spirit and scope of the present invention.All such modifications are intended to be within the scope of the claimsappended hereto.

1-74. (canceled)
 75. A method of synthesizing a non-peptide smallmolecule comprising reacting a first ligand with a second ligand,wherein (a) the first ligand has inherent binding affinity for a firstsite of interest on a Target Biological Molecule (TBM) having a reactivenucleophile; and (b) the second ligand has been identified from among alibrary of ligand candidates to have inherent binding affinity for asecond site of interest on said TBM by screening said library with acomplex formed between a reactive derivative of said first ligand andthe nucleophile of said TBM resulting in the formation of anirreversible covalent bond between said complex and said second ligand:76. The method of claim 75 wherein said first ligand has been identifiedfrom among a library of ligand candidates by screening said library withsaid TBM under conditions resulting in the formation of a reversiblecovalent bond between said nuclephile and said first ligand
 77. Themethod of claim 75 wherein said nucleophile is selected from the groupconsisting of thiol, protected thiol, reversible disulfide, hydroxyl,protected hydroxyl, amino, protected amino, carboxyl and protectedcarboxyl groups.
 78. The method of claim 75 wherein said first andsecond ligands are covalently linked to one another through a disulfidebond.
 79. The method of claim 75 wherein said first and second ligandsare directly fused to one another.
 80. The method of claim 75 whereinsaid small molecule consists essentially of said first and secondligands, covalently linked through a disulfide bond.
 81. The method ofclaim 78 further comprising the step of synthesizing derivatives of saidsmall molecule.
 82. The method of claim 81 wherein the disulfide bondcovalently linking said first and second ligands to one another isreplaced by a different covalent linkage.
 83. The method of claim 82wherein the different linkage is a straight chain, branched chain oraromatic linker.
 84. The method of claim 83 wherein the differentlinkage is a straight chain linker of at least two atoms in length. 85.The method of claim 83 wherein the different linkage is a straight chainlinker of at least four atoms in length.
 86. The method of claim 83wherein the different linkage is a straight chain linker of at least 12atoms in length.